Analysis of gene expression profiles in insulin-sensitive tissues from pre-diabetic and diabetic Zucker diabetic fatty rats

in Journal of Molecular Endocrinology
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  • 1 Division of Metabolic Diseases, Department of Biomedical Sciences, National Institute of Health, 5 Nokbun-dong, Eunpyung-gu, Seoul, 122-701, South Korea
  • 2 1Research and Development, GenomicTree, Inc., 461-6 Jonmin-dong, Yusong-gu, Taejon 305-811, South Korea

Insulin resistance occurs early in the disease process, preceding the development of type 2 diabetes. Therefore, the identification of molecules that contribute to insulin resistance and leading up to type 2 diabetes is important to elucidate the molecular pathogenesis of the disease. To this end, we characterized gene expression profiles from insulin-sensitive tissues, including adipose tissue, skeletal muscle, and liver tissue of Zucker diabetic fatty (ZDF) rats, a well characterized type 2 diabetes animal model. Gene expression profiles from ZDF rats at 6 weeks (pre-diabetes), 12 weeks (diabetes), and 20 weeks (late-stage diabetes) were compared with age- and sex-matched Zucker lean control (ZLC) rats using 5000 cDNA chips. Differentially regulated genes demonstrating > 1.3-fold change at age were identified and categorized through hierarchical clustering analysis. Our results showed that while expression of lipolytic genes was elevated in adipose tissue of diabetic ZDF rats at 12 weeks of age, expression of lipogenic genes was decreased in liver but increased in skeletal muscle of 12 week old diabetic ZDF rats.

These results suggest that impairment of hepatic lipogenesis accompanied with the reduced lipogenesis of adipose tissue may contribute to development of diabetes in ZDF rats by increasing lipogenesis in skeletal muscle. Moreover, expression of antioxidant defense genes was decreased in the liver of 12-week old diabetic ZDF rats as well as in the adipose tissue of ZDF rats both at 6 and 12 weeks of age. Cytochrome P450 (CYP) genes were also significantly reduced in 12 week old diabetic liver of ZDF rats. Genes involved in glucose utilization were downregulated in skeletal muscle of diabetic ZDF rats, and the hepatic gluconeogenic gene was upregulated in diabetic ZDF rats. Genes commonly expressed in all three tissue types were also observed. These profilings might provide better fundamental understanding of insulin resistance and development of type 2 diabetes.

Abstract

Insulin resistance occurs early in the disease process, preceding the development of type 2 diabetes. Therefore, the identification of molecules that contribute to insulin resistance and leading up to type 2 diabetes is important to elucidate the molecular pathogenesis of the disease. To this end, we characterized gene expression profiles from insulin-sensitive tissues, including adipose tissue, skeletal muscle, and liver tissue of Zucker diabetic fatty (ZDF) rats, a well characterized type 2 diabetes animal model. Gene expression profiles from ZDF rats at 6 weeks (pre-diabetes), 12 weeks (diabetes), and 20 weeks (late-stage diabetes) were compared with age- and sex-matched Zucker lean control (ZLC) rats using 5000 cDNA chips. Differentially regulated genes demonstrating > 1.3-fold change at age were identified and categorized through hierarchical clustering analysis. Our results showed that while expression of lipolytic genes was elevated in adipose tissue of diabetic ZDF rats at 12 weeks of age, expression of lipogenic genes was decreased in liver but increased in skeletal muscle of 12 week old diabetic ZDF rats.

These results suggest that impairment of hepatic lipogenesis accompanied with the reduced lipogenesis of adipose tissue may contribute to development of diabetes in ZDF rats by increasing lipogenesis in skeletal muscle. Moreover, expression of antioxidant defense genes was decreased in the liver of 12-week old diabetic ZDF rats as well as in the adipose tissue of ZDF rats both at 6 and 12 weeks of age. Cytochrome P450 (CYP) genes were also significantly reduced in 12 week old diabetic liver of ZDF rats. Genes involved in glucose utilization were downregulated in skeletal muscle of diabetic ZDF rats, and the hepatic gluconeogenic gene was upregulated in diabetic ZDF rats. Genes commonly expressed in all three tissue types were also observed. These profilings might provide better fundamental understanding of insulin resistance and development of type 2 diabetes.

Introduction

Type 2 diabetes mellitus is a common metabolic disease involving abnormal carbohydrate regulation and lipid metabolism by insulin (Saltiel 2001). Insulin resistance, characterized as decreased insulin action on glucose uptake and metabolism, occurs prior to the onset of type 2 diabetes and plays a major role in its development. Contributing to insulin resistance, obesity is known as a strong risk factor for type 2 diabetes. However, obesity alone does not account for disease development, as only a small fraction of obese people develop diabetes, with most being able to maintain a euglycemic state despite also being insulin resistant. Since it is likely that the development of type 2 diabetes is determined in part by genetic factors, understanding differences in gene expression of insulin-sensitive tissues from animals in different progressive stages of diabetes development (e.g. insulin resistant pre-diabetes and diabetes) may help uncover the underlying molecular mechanisms involved in disease progression.

A recent advancement in genomic research, microarray technology allows for the expression of thousands of genes to be quickly and easily monitored in parallel. Global gene expression, or transcriptional profiling, has been used to identify molecular markers for various pathological states and can be used to generate novel hypotheses to characterize different disease states. Studies using microarrays have been performed to identify candidate genes related to insulin resistance and diabetes in animal and human models of diabetes (Yang et al. 2002, Sreekumar et al. 2002, Lan et al. 2003, Rome et al. 2003, Mootha et al. 2003). However, until recently, there was no full set of alterations in gene expression that may possibly be involved in the disease pathology of type 2 diabetes.

A well-characterized model of obesity and type 2 diabetes, the Zucker diabetic fatty (ZDF) rat, has a point mutation in the leptin receptor that leads to impairment of the signaling capabilities of this receptor. The ZDF rat develops an age-dependent diabetes phenotype, with onset of obesity at 5–7 weeks of age accompanied by a metabolic state of early diabetes mellitus with hyperinsulinemia and insulin resistance, but with euglycemia. The full syndrome of diabetes develops at 10–12 weeks with hyperglycemia. The diabetic pathogenetic features manifested in this animal model are in many ways reminiscent of the pathogenesis of type 2 diabetes in humans. Therefore, the ZDF animal model is ideal for identifying molecules contributing to insulin resistance related to obesity and to eludiate the molecular pathogenesis of type 2 diabetes.

In this study, using cDNA microarrays, global gene expression in adipose tissue, skeletal muscle, and liver tissue were profiled from 6-week old pre-diabetic ZDF rats, 12-week old diabetic ZDF rats and 20-week old late-stage diabetic ZDF rats compared with age and sex-matched Zucker lean control (ZLC) rats. This approach provided several candidate genes and possible molecular mechanisms responsible for insulin resistance and progression to type 2 diabetes.

Materials and methods

Animals

Male Zucker diabetic fatty (ZDF/Gmi-fa/fa) rats (6, 12 or 20 weeks old) and their sex- and age-matched Zucker lean control (ZLC/Gmi-+/fa) rats were purchased from Genetic Models (GMI, Indianapolis, IA, USA). Animals were maintained on a commercial chow diet ad libitum before being killed. Body weights and blood glucose levels for the 6- and 12-week old ZDF rats were pre-diabetic and diabetic stage-appropriate, respectively. The rats were sacrificed by cervical dislocation, and the epididymal fat, skeletal muscle of the femoral region, and liver were removed and subjected to total RNA extraction.

RNA preparation and labeling

Total RNA was isolated using the TRIzol reagent (GibcoBRL, Invitrogen Corporation, Carlsbad, CA, USA). The cDNA were labeled with the 3DNA Array kit (Genisphere, Hatfield, PA, USA) according to the manufacturer’s protocol and recommendations. The cDNA of ZDF rats was labeled with Cy5, and cDNA of each paired control ZLC rat was labeled with Cy3. Briefly, total RNA (20 μg) and the Cy3 or Cy5 capture sequence primer were solubilized in 20 μl of diethyl pyrocarbonate (DEPC)-treated water and incubated for 10 min at 80 °C. The RNA mixture was then transferred on to ice and the RNase inhibitor was added. An equal volume of reaction mixture containing 5X first-strand buffer, 100 mM DTT, 10 mM dNTP mix and 200 units of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was added and the reaction was incubated for 2 h at 42 °C. The reaction was terminated by adding 0.5 M NaOH/50 mM EDTA and incubated at 65 °C for 10 min. Finally, the reaction was neutralized with 1 M Tris–HCl, pH 7.5. The cDNA samples were concentrated by centrifugation using Centricon filters (Millipore, Billerica, MA, USA).

cDNA microarray analysis

The cDNA microarray analysis was performed on rat expression glass microarrays (Genomic Tree, Taejon, Korea), which were spotted with 5000 cDNA of known rat genes and expressed sequence tags (ESTs). A total of 36 microarray slides (3 ages × 3 tissue types × 4 replicates) were used to monitor gene expression levels. Hybridization and washing of the microarrays were carried out according to the manufacturer’s instructions. In brief, cDNA probe solutions containing 2X hybridization buffer and Array 50 dT blocker were applied to the microarrays, which were then covered with a spaced glass coverslip and placed in a humidified chamber at 65 °C for 16 h. Then, the microarrays were sequentially washed for 15 min each, once at 65 °C in 2 × SSC and 0.2% SDS, once in 2 × SSC at room temperature, and once in 0.2 × SSC at room temperature, and then washed for 2 min at room temperature in 95% ethanol to fix the cDNA molecules to the probes. Then, the microarrays were centrifuged for 2 min at 1000 r.p.m. to dry the slide. In preparation for 3DNA hybridization, the 3DNA Capture Reagent and the anti-fade reagent were added to the hybridization buffer. The prepared hybridization buffer was added to the arrays and incubated for 2 h in a dark humidified chamber at 65 °C. The microarrays were again sequentially washed and dried.

Scanning and data analysis

The hybridization images were quantitated by GenePix Pro 4.0 (Axon Instruments, Union City, CA, USA). The average fluorescence intensity for each spot was calculated and local background was subtracted. All data normalization and statistical analysis were performed using GeneSpring 6.1 (Silicon Genetics, Redwood, CA, USA). Genes were filtered according to their intensity in the control channel based on the two-component model for estimating variation from control strength (Rocke & Durbin 2001). Intensity-dependent normalization (LOWESS) was performed, where the ratio was reduced to the residual of the LOWESS fit of the intensity vs ratio curve. In addition, to adjust the data from three different ages, we performed median centering by dividing each gene’s signal ratio by the median of all the signal ratios for that particular gene. The values of fold change were calculated by dividing the median of normalized signal channel intensity (Cy5) by the median of normalized control channel intensity (Cy3). The ANOVA test (parametric) was performed at P values < 0.05 to find genes that differentially expressed across conditions. Unsupervised hierarchical cluster analysis was performed by similarity measurements based on Pearson correlations around zero.

Real-time reverse transcription PCR (RT-PCR)

For real-time RT-PCR, cDNA was prepared from 3 μg of total RNA from ZDF or ZLC rats using random hexamers and the Moloney murine leukemia viral reverse transcriptase (Promega, Madison, WI, USA) in a final reaction volume of 20 μl. The resulting cDNA was analyzed by real-time PCR using SYBR Green PCR core reagents (PE Biosystems, Warrington, UK). PCR was performed in an ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA, USA) and each gene-specific primer set was used at concentrations of 150 nM in a final volume of 25 μl. The accuracy of the PCR products was verified by agarose gel electrophoresis. Quantification of a given gene, expressed as relative mRNA level compared with control levels, was calculated after normalization to 18S rRNA and using the ΔΔCT formula as described by Perkin Elmer. Individual CT values were calculated as means of duplicate or triplicate measurements.

Western blotting

Skeletal muscle or liver tissues from ZDF (6 and 12 weeks) and ZLC (6 and 12 weeks) rats were solubilized in RIPA buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin) for 30 min on ice. The lysates were clarified by centrifugation and separated by SDS-PAGE (10%), transferred onto nitrocellulose filter. The filters were probed with SREBP-1 (H-160) or PPARγ (H-100) antibody purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) for 16 h. Membranes were washed and incubated with a 1:4000 dilution of horseradish peroxidase conjugated secondary antibodies. Protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Results

Overview of gene expression profiles in ZDF rats

Six-week old ZDF male rats, together with 6-week old ZLC rats serving as controls, were used to study pre-diabetic changes, whereas 12-week old ZDF male rats and age-matched ZLC rats were used to study diabetic changes, and 20-week old ZDF male rats were used to study late-stage diabetic changes. Microarrays were carried out to study gene expression in adipose tissue, skeletal muscle, and liver tissue from ZDF and ZLC rats using cDNA chips containing 5000 rat genes. In this study, four ZDF and ZLC rats at each age were used. Total RNA was isolated from three different tissues from ZDF or ZLC rats of corresponding ages. Four pairs of each Cy5-labeled ZDF and Cy3-labeled ZLC rat cDNA samples were applied to a cDNA chip respectively, which was essentially equivalent as applied in four replications. After performing intensiy-dependent normalization on spots of 12 slides of each tissue (3 ages × 4 replicates), fold-changes were determined by dividing the median normalized signal channel intensity by the median normalized control channel intensity. Differentially expressed genes (P < 0.05) as determined by ANOVA and with about a 1.3 fold-change in expression levels were selected. Differences observed at 6 weeks, which corresponds to the period preceding diabetes onset, were considered to be involved in causing type 2 diabetes, whereas those seen at 12 weeks were considered likely to be involved in the progression of type 2 diabetes. There were 381 genes found in adipose tissue, 319 genes from skeletal muscle, and 528 genes from liver that exhibits significant changes of expression during type 2 diabetes. These genes were further analyzed using hierarchical clustering to group genes with similar changes in expression profiles. As shown in Fig. 1, hierachical clustering revealed that four replicates showed little variance of expression in each age for the three tissues, indicative of high reliability of chip data. Furthermore, the differentially expressed genes in adipose, skeletal muscle and liver were clustered into 4, 4, and 5 groups, respectively. Many of the differentially expressed genes in adipose tissue had a similar expression pattern in 6-week old pre-diabetic ZDF rats and 12-week old diabetic ZDF rats, whereas many genes in the liver and skeletal muscle had reverse expression patterns at 6 and 12 weeks of ages. Namely, many of the genes that were up- or downregulated in the liver and skeletal muscle of pre-diabetic ZDF rats became down-or upregulated reversely in diabetic 12-week old ZDF rats. Therefore, genes whose expression was differentially regulated from 6 to 12 weeks may contribute to the development of type 2 diabetes, along with up- or downregulated genes found in the tissues of diabetes-stage ZDF rats. Tables 1 to 3 list the representative genes in each cluster along with the putative biological functions of their encoded proteins, as found in searches of the public database. Through quantitative real-time RT-PCR, we confirmed expression levels of genes selected on the basis of biological interest or ranking P value in each tissue from cross counterpart animals. As shown in Fig. 2, the expression of genes selected in skeletal muscle and liver was concordant with microarray data, although value differences did not match exactly.

Differentially expressed genes in insulin sensitive tissues of ZDF rats

In the adipose tissue of ZDF rats, the expresson of serine (or cysteine) proteinase inhibitor 1 (PAI-1) in cluster 1, as a known molecular marker of obesity linked to type 2 diabetes, was increased in diabetic ZDF rats at 12 weeks, consistent with previous reports showing that elevated expression levels of PAI-1 was associated with insulin resistance related to obesity and type 2 diabetes (Bastard et al. 2000). Additionally, PAI has been reported to inhibit insulin signaling by competing with αvβ3 integrin for vitronectin binding (Lopez-Alemany et al. 2003), which may explain its role in the development of type 2 diabetes. Furthermore, the expression level of angiotensinogen (AGT) which is involved in blood pressure regulation and adipocyte differentiation, was downregulated in 6-week old pre-diabetic ZDF rats, but upregulated in 12-week old diabetic ZDF rats. Previously, the expression level of AGT in ZDF rats has been reported to be up- or downregulated depending on the age of animals used (Jones et al. 1997, Hainault et al. 2002). Metallothionein 3, identified in cluster 4, which may have an antioxidant role as a metal-binding and stress-response protein, was downregulated in both pre-diabetic and diabetic ZDF rats. The expression of vitamin D binding protein (DBP), essential for cellular endocytosis and intracellular metabolism of vitamin D, also was decreased in both pre-diabetic and diabetic ZDF rats. Vitamin D is necessary for maintaining insulin secretion and normal glucose tolerance and influences insulin sensitivity. DBP gene variants have also been shown to be predisposed to type 2 diabetes mellitus (Malecki et al. 2002). Therefore, the decreased expression of DBP is expected to induce insulin resistance. The expression of ATP citrate lyase and stearoyl-coenzyme A desaturase 2 in cluster 2 and 3 respectively, which are involved in triglyceride synthesis, was found to be decreased in 12-week old ZDF rats, whereas lipolytic genes such as hormone sensitive lipase and fatty acid binding proteins in cluster 1 were upregulated at 12 weeks.

In the skeletal muscle of ZDF rats, the expression of pyruvate dehydrogenase and NAD(P)H dehydrogenease for glucose oxidation identified in cluster 1 and 4 respectively, were decreased in 12-week old diabetic ZDF rats. Transgellin 3, whose function is related to muscle development, was also downregulated at 12 weeks, suggesting that this gene may play an important role in insulin resistance and type 2 diabetes since insulin resistance is associated with dysregulated myogenic development. The expression of SorCS2 and sortilin genes in cluster 2 and 3 was decreased in 6-week old pre-diabetic ZDF rats. These genes belong to the novel type I transmembrane receptor family containing a common Vps10 domain, a sorting receptor for the carboxypeptidase Y. It has been suggested that sortilin has a cytoplasmic tail sequence homology between sortilin and insulin like growth factor II (Mazella 2001), and co-localizes in intracellular vesicles containing glucose transporter 4 (GLUT4) and translocates into the plasma membrane in response to insulin (Morris et al. 1998). SorCS2 has an amino acid sequence similar with sortilin except for the presence of isoleucine/valine rich repeats (Hermey et al. 1999). Therefore, the down-regulation of Vps10 domain-containing proteins might influence the translocation of GLUT4 in skeletal muscle. Parvalumin and triadin in cluster 1, which play an important role in regulating intracellular calcium concentration through their buffering capacity as high-affinity cytosolic calcium binding proteins (CBPs), were significantly downregulated in ZDF rats 12-weeks old. Regulation of intracellular calcium plays a key role in obesity, insulin resistance, and hypertension. Calcium has been shown to regulate insulin signaling, as increasing levels of calcium impairs insulin signaling, possibly through inhibition of insulin-regulated dephosphorylation, resulting in insulin resistance. Therefore, the reduced expression of CBP in diabetic-stage rats may cause an increase in intracellular calcium and lead to insulin resistance.

In the livers of ZDF rats, glucose-6-phosphatase in cluster 3, involved in gluconeogenesis, was upregulated at 12 weeks, which could contribute to hyperglycemia in ZDF rats. Expression of the mitogen activated protein kinase (MAPK) genes, including stress activated protein kinase 2 and MAPK3 (ERK1), identified in cluster 2, was increased at 6 weeks, and the regulator of G-protein signaling 12 was increased at 12 weeks.

Decreased lipogenic gene expression in diabetic liver, but increased lipogenic gene expression in diabetic skeletal muscle of 12-week old ZDF rats

We found that lipogenic genes, including ATP citrate lyase and fatty acid coenzyme A ligase (FACL), were downregulated in the livers of 12-week old diabetic ZDF rats. In order to further confirm the reduced expression of lipogenic genes in the livers of diabetic ZDF rats, we measured the expression of other lipogenic genes including fatty acid binding protein (aP2), peroxisome proliferator activated receptor gamma (PPARγ), and SREBP1c with genes observed in this study at 6 and 12 weeks of age using real-time RT-PCR. As shown in Fig. 3A, expression levels of aP2, PPARγ, and SREBP1c genes were significantly lower in 12-week old ZDF rats compared with 12-week old ZLC rats, consistent with microarray data. The data also showed that the expression of aP2 and PPARγ was increased from 6 to 12 weeks in both strains, but the increase in ZDF rats was much smaller than in ZLC rats. The expression of SREBP1c and FACL2 was increased from 6 weeks to 12 weeks in ZLC rats, whereas the expression was decreased in ZDF rats. Protein expression levels of PPARγ and SREBP1 were consistent with the results of real-time RT-PCR (Fig. 3C).

In contrast to the decreased lipogenic gene expression found in diabetic livers of ZDF rats, the expression of the lipogenic genes, fatty acid coenzyme A ligase, fatty acid synthase, and ATP citrate lyase, were increased in the skeletal muscle of 12-week old diabetic ZDF rats. Expression of other lipogenic genes including PPARγ, SREBP1c, and stearoyl-coenzyme A desaturase 1 (SCD1) were also measured in the skeletal muscle at 6 and 12 weeks of age with real-time RT-PCR. As shown in Fig. 3B, the expression of SCD1 and FACL2 was significantly higher in the skeletal muscle of 12-week old ZDF rats compared with ZLC rats of the same age. Even though expression of PPARγ and SREBP1c was not shown to be significantly different between ZLC and ZDF rats at 12 weeks of age, the expression of these genes in diabetic ZDF rats was increased from 6 weeks to 12 weeks in contrast to decreases observed in ZLC rats. The protein levels of PPARγ and SREBP1 were also consistent with transcription levels (Fig. 3C).

Reduced expression of antioxidant genes and CYP450 genes in diabetic livers from 12-week old ZDF rats

Our microarray data also revealed that antioxidant genes, such as hemopexin, glutathione peroxidase 1, superoxide dismutase 1, and glutaredoxin 1 were downregulated in the liver of ZDF rats at 12 weeks of age. Furthermore, the expression of cytochrome P450 (CYP) genes, including CYP4A3, CYP1A2, CYP3A9, and CYP2C39 was also significantly decreased at 12 weeks, although the expression of these genes was increased at 6 weeks. These data strongly suggest that the liver of diabetic ZDF rats at 12 weeks of age has an impaired capacity to manage oxidative stress and some xenobiotics. Therefore, we verified the expression of gluthathione-related genes and CYP 450 genes in both animals at 6 and 12 weeks of ages with the real-time RT-PCR. As shown in Fig. 4, the expression of glutathione peroxidase 1, superoxide dismutase, and glutaredoxin 1 was decreased in the liver of 12-week old diabetic ZDF rats compared with ZLC rats of the same age, consistent with microarray data. Moreover, the expression of these genes was significantly decreased from 6 weeks to 12 weeks in ZDF rats, whereas the expression was increased in ZLC rats. The expression of CYP4A3, 1A2, 3A9 and 2C39, as shown in Fig. 5, was also decreased in 12-week old ZDF rats.

Common genes expressed concurrently in insulin sensitive tissues of ZDF rats

Several common genes expressed concurrently in insulin sensitive tissues of ZDF rats were observed (Table 4). Of them, calpain 4 was downregulated in skeletal muscle and liver of 12-week old rats but in all three tissues of 20-week old diabetic ZDF rats. Annexin1, cathespsin B, and vimentin were upregulated in both pre-diabetic muscle and liver tissue from ZDF rats, but their expression was decreased in diabetic-stage animals. Lumican and enolase 2 were also upregulated in both pre-diabetic adipose and skeletal muscle in ZDF rats, whereas collapsing response mediator protein 1 was significantly downregulated in both pre-diabetic tissue types. Growth associated protein 43, collagen (type 1 α1), and transgelin 3 were downregulated in both diabetic adipose and skeletal muscle although the expression was increased in pre-diabetic rats. The expression of protein phosphatase 5 was increased in both diabetic adipose and pre-diabetic liver tissues.

Discussion

Using microarray technology, it is now possible to examine the transcriptional profile of several thousand genes simultaneously to decipher the pathophysiology of the disease at molecular level. In this study, we assessed the expression profiles of 5000 genes in insulin sensitive tissues from 6-week old pre-diabetic ZDF rats, 12-week old diabetic ZDF rats, and 20-week old late-stage diabetic ZDF rats for a better fundamental understanding of insulin resistance and type 2 diabetes, Collectively, our results suggest a pathophysiological cascade of gene expression in type 2 diabetes in the ZDF model.

Lipolytic gene expression was increased in the adipose tissue of 12-week old diabetic ZDF rats. Increased free fatty acids (FFA) released by adipocytes is a key feature in type 2 diabetes. Since the balance between cellular triglyceride synthesis, FFA re-esterification, and triglyceride hydrolysis determines the amount of FFA released by adipocytes, aberrations in these cellular pathways may contribute to increased FFA production in adipose tissue. Elevated plasma concentrations of FFA can cause insulin resistance by impairing the ability of insulin to stimulate muscle glucose uptake and to inhibit hepatic glucose production (Lewis et al. 2002). Lipolysis in adipose tissue is mediated, in part, via interaction of fatty acid binding protein (FABP) with hormone sensitive lipase (Lewis et al. 2002). In our data, ATP citrate lyase and stearoyl-coenzyme A desaturase 2, involved in triglyceride synthesis, were downregulated in adipose tissue of 12-week old ZDF rats, whereas lipolytic genes such as hormone sensitive lipase and FABP were upregulated at 12 weeks. This imbalance may increase the release of FFA from adipose tissue in diabetic ZDF rats and could lead to further insulin resistance and development of type 2 diabetes.

While lypolytic gene expression was increased in the adipose tissues of 12-week old diabetic ZDF rats, the expression of genes involved in lipogenesis was decreased in the liver as well as in the adipose tissue of diabetic ZDF rats at 12 weeks. Reportedly, adipose function, reflected in the expression of lipogenic genes, deteriorates with prolonged obesity (Nadler et al. 2000) and reduced lipogenic adipocytes are associated with increased hepatic lipogenesis, which may be a shift in the lipogenic burden from adipocytes to other organs such as the liver (Diraison et al. 2002). However, a failure to increase hepatic lipogenesis in obesity has been shown to contribute to the development of type 2 diabetes (Lan et al. 2003). Moreover, increased hepatic lipogenesis is also associated with reduced risk of diabetes in a lipoatrophic mouse model (Colombo et al. 2003). In our study, the expression of ATP citrate lyase and fatty acid coenzyme A ligase for lipogenesis was significantly downregulated in the liver of 12-week old diabetic ZDF rats although the expression was increased in 6-week old pre-diabetic rats. The expression of other lipogenic genes such as aP2, PPARγ, and SREBP1c also decreased significantly in 12-week old ZDF rats compared with 12-week old ZLC rats, which is shown in Fig. 3A and 3C. Previous reports also showed that lipogenic gene expression in the livers of diabetes-susceptible BTBR-ob/ob was decreased compared with diabetes-resistant B6 ob/ob animals, in addition to a dramatic decrease of the expression from 6 weeks (pre-diabetes) to 14 weeks (diabetes) in BTBR-ob/ob (Lan et al. 2003). These results suggested that the livers of ZLC rats maintain a high level of hepatic lipogenesis at 12 weeks, whereas lipogenesis in the diabetic livers of ZDF rats was impaired in the transition from the pre-diabetes to diabetes stage. Therefore, the impairment of hepatic lipogenesis in diabetic ZDF rats may contribute to the progression of type 2 diabetes by increasing glucose production and shifting lipogenic burdens to skeletal muscle with deteriorated adipose function.

With impairment of lipogenesis in the livers of 12-week old ZDF rats, we observed an increase in the expression of lipogenic genes, including fatty acid synthase, ATP citrate lyase, and fatty acid coenzyme A ligase in the skeletal muscle of diabetic ZDF rats at 12 weeks of age. Expression of other lipogenic genes, including PPARγ, SREBP1c, and SCD1 also was increased in the skeletal muscle of 12-week old ZDF rats compared with ZLC rats of the same age as shown in Fig. 3B and 3C. Moreover, expression of these lipogenic genes was increased from 6 weeks to 12 weeks, whereas the expression was decreased in ZLC rats. Potentially, these increases could contribute to muscular triglyceride accumulation, leading to insulin resistance or type 2 diabetes. With intramuscular lipid accumulation, lipid intermediates such as long-chain fatty acyl CoA, diacylglycerol, and ceramide are increased. Increasing lipid intermediates inhibit insulin signaling and insulin-mediated glucose uptake in skeletal muscle (Petersen & Shulman 2002, Schmitz-Peiffer 2002, Lam et al. 2003). Therefore, upregulated lipogenesis in the skeletal muscle of diabetic stage animals could contribute to the development of obesity-related diabetes.

Increased hepatic gluconeogenic gene expresson in the livers of 12-week old diabetic ZDF rats was observed, as well as reduced gene expression of glucose oxidation in the skeletal muscle and adipose tissue of ZDF rats at 12 weeks. Expression of glucose-6-phosphatase was increased in the liver of ZDF rats at 12 weeks of age, resulting in hyperglycemia. The committed step for glucose oxidation is the conversion of pyruvate to acetyl CoA in the mitochondria by the pyruvate dehydrogenase complex, which then enters the TCA cycle. In our study, expression of pyruvate dehydrogenase (PDX1) and NADP(H) dehydrogenase was decreased in the skeletal muscle of diabetic ZDF rats, and voltage-dependent anion channel for oxidative phosphorylation was also decreased in the adipose tissue of diabetic ZDF rats. These results suggest that oxidative metabolism is decreased in both skeletal muscle and adipose tissue of diabetic ZDF rats at 12 weeks of age.

The expression of antioxidant genes was downregulated in adipose and liver tissues of 12-week old diabetic ZDF rats. More recently, studies have linked reactive oxygen species (ROS) production and oxidative stress to insulin resistance (Evans et al. 2002). In vitro, ROS and oxidative stress led to the activation of multiple serine kinase cascades, which phosphorylates insulin-signaling substrates including the insulin receptor (IR) and insulin receptor substrate (IRS)-1, and inhibits tyrosine phosphorylation (Evans et al. 2002). Therefore, antioxidants could prevent the activation of these serine kinases induced by ROS and oxidative stress. In our study, metallothionein 3, which plays a role as an antioxidant protecting ROS, as well as a detoxifying antidote for metals, was downregulated in the adipose tissues of ZDF rats at both ages. Hemopexin (Hpx), which acts as an antioxidant through its strong heme binding, was also downregulated in liver of diabetic ZDF rats progressing from pre-diabetes to diabetes. Importantly, the expression of glutathione-related defense genes including glutathione peroxidase 1, superoxide dismutase 1, and glutaredoxin 1 was increased in the livers of 6-week old pre-diabetic ZDF rats which may be responsible for defenses against oxidative stress, but was significantly reduced at 12 weeks (diabetes stage). We verified the expression of these genes in both animals at each age with real-time RT-PCR. As shown in Fig. 4, expression levels in ZLC rats were increased from 6 weeks to 12 weeks, whereas expression in ZDF rats was significantly decreased. Therefore, the reduced expression of these antioxidant genes in diabetic livers may partly account for insulin resistance and development of type 2 diabetes.

Although the expression of cytochrome P450 (CYP) was increased in 6-week old pre-diabetic ZDF rats, CYP450 expression was significantly reduced in the livers of 12-week old diabetic ZDF rats. Cytochrome P450 constitutes a superfamily of hemeproteins that play important roles in the detoxification of numerous xenobiotics, as well as endogeneous compounds including steroids, fatty acids, and prostaglandins. In various disease states, including diabetes, obesity, and inflammation and infection, the expression of hepatic P450 in the liver changes markedly. In streptozotocin-induced diabetes, hepatic expression of CYP2B, CYP2E1, and CYP1A2 was increased, but expression of CYP2C11, CYP2C13, CYP2A2, and CYP3A2 was decreased (Thummel & Schenkman 1990). In New Zealand obese mice, which exhibit a polygenic syndrome of obesity, insulin resistance, dyslipidemia, and hypertension, the expression of CYP2B9, CYP3A16, and CYP4A14 was increased markedly in livers from diabetic mice, but only slightly increased in insulin resistant mice. In contrast, expression of CYP2C22, CYP2C29 and CYP2C40 was reduced in diabetic, but not affected in insulin resistant mice. Furthermore, expression of CYP1A2 and CYP7B1 was reduced in both diabetic and insulin resistant mice (Pass et al. 2002). In our present study, expression of CYP4A3, CYP3A9, CYP2C39, and CYP1A2 was increased in the livers of pre-diabetic ZDF rats, but was significantly reduced in diabetic livers, which was confirmed by real-time RT-PCR (Fig. 5). Although these alterations correlated with changes in serum free fatty acid levels and seem to be mediated by PPAR-α, the reduction of these CYP enzymes in diabetic livers of ZDF rats may contribute to the development of type 2 diabetes due to a reduced capacity for xenobiotic detoxification.

Analyzing microarray data, we found that the expresson of MAP serine/threonine kinase pathway genes was up- or downregulated depending on the tissue type. Stress activated protein kinase (SAPK) alpha 2 (referred to as JNK/SAPK) and extracellular signal regulated kinase1 (ERK-1) were upregulated in the livers of 6-week old pre-diabetic ZDF rats, but mitogen activated protein kinase kinase 6 (MKK6), which activates p38 MAPK, was downregulated in the skeletal muscle of ZDF rats at 12 weeks of age. It was reported that constitutive activation of MEK1–ERK down-regulates the expression of GLUT4, IR, IRS-1 and IRS-2, resulting in marked impairment of insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2, docking of the p85α regulatory subunit of PI3K, and PI3K activation (Hirosumi et al. 2002). Furthermore, the activation of MKK7-JNK pathway also suppressed tyrosine phosphorylation of IRS-1 or IRS-2, which in turn, suppressed activation of PI3K and Akt. It was also reported that JNK/SAPK activity is abnormally elevated in obesity (Hirosumi et al. 2002). Taken together, activation of ERK, JNK can induce insulin resistance in pre-diabetic livers. Previous reports demonstrated that the p38 MAPK inhibitor, SB203580, prevented insulin-stimulated glucose uptake in 3T3-L1 adipocyte and L6 muscle cells (Sweeney et al. 1999) and that alpha lipoic acid activated glucose uptake via a p38 MAPK-dependent pathway (Konrad et al. 2001). Therefore, downregulation of MKK6, upstream of p38 MAPK, may lead to decreased insulin-stimulated glucose transporter in diabetic skeletal muscle.

We also observed several common genes regulated differentially in insulin sensitive tissues including annexin, regulator of G-protein signaling (RGS), and calpain 4. Annexin 1 was upregulated in the skeletal muscle and livers of pre-diabetic ZDF rats. The annexin superfamily, which consists of at least 11 different, abundant, and ubiquitously expressed proteins, reportedly play important roles in the regulation of insulin secretion and the effects of insulin on its target tissues by inhibition of IR tyrosine phosphorylation (Melki et al. 1994). Furthermore, it was reported that the annexin 1 gene harbors variants that increase the risk of type 2 diabetes (Lindgren et al. 2001). All together, upregulation of annexin 1 in pre-diabetic skeletal muscle and liver tissue may induce insulin resistance and contribute to the development of type 2 diabetes. Additionally, expression of the regulator of G-protein signaling (RGS) gene, which is essential for catecholamine and insulin action, was increased in pre-diabetic skeletal muscle and diabetic liver tissue in ZDF rats. The G protein signaling pathway is regulated by the interplay between receptor-catalyzed activation and inhibitory RGS proteins. RGS proteins acts like a GTPase-activating protein (GAP) specific for Gi and Gq class G protein alpha subunit, and plays a crucial role in processes responsible for shutting off G-protein-mediated cell responses in eukaryotes (Ishii & Kurachi 2003). Recent observations have found possible linkages between heterotrimeric G-proteins and insulin signaling (Luft 1997). Reportedly, the G alpha-q/11 protein is required for insulin-induced glucose transport in 3T3-L1 adipocyte in PI3-kinase dependent or independent signal transduction pathways (Imamura et al. 1999). Therefore, the upregulation of RGS in insulin sensitive tissues could attenuate insulin stimulated GLUT4 translocation by inhibiting signaling by GTP binding protein, and lead to insulin resistance in the pre-diabetic and diabetic stages. Another cysteine proteinase, calpain 4, was downregulated in the skeletal muscle and liver of 12-week old diabetic ZDF rats, although the expression was increased in 6-week old pre-diabetic ZDF rats. Recently, one member of the calpain family, calpain 10, was identified by positional cloning as a susceptibility gene for type 2 diabetes. Genetic studies have shown that variations in the calpain gene are associated with type 2 diabetes (Horikawa et al. 2000). Furthermore, low levels of calpain 10 mRNA in skeletal muscle has also been reportedly associated with insulin resistance (Baier et al. 2000). Treatment of muscle strips with the calpain inhibitor, ALLM and E-64-d resulted in a significant reduction in the rate of insulin-stimulated glucose uptake and the incorporation of glucose into glycogen (Sreenan et al. 2001). Therefore, the reduction of calpain 4 gene expression from pre-diabetes to diabetes in insulin sensitive tissues may also attenuate the insulin-stimulated glucose uptake and play an important role in the development of type 2 diabetes.

In summary, we assessed global transcriptional profiles of insulin sensitive tissues of ZDF male rats at a pre-diabetic and diabetic stage. This approach, coupled with current metabolic knowledge, provided several candidate genes and molecular pathology of insulin resistance and the development of type 2 diabetes.

Table 1

Genes differentially expressed in adipose tissue of ZDF rats

Normalized expression (ZDF/ZLC)
Gene Name6W12W20WFunction
Expression ratios are shown by log2 scale of normalized mean signal intensities of ZDF rat genes to those of ZLC. ND denotes “non-detectable or low signal-to-noise ratio”. W, weeks old.
Accession
 Cluster 1
AA901043EST0.001.75−1.12Unclassified
AA900150NAD+-specific isocitrate dehydrogenase b subunit0.071.52−1.04Metabolism
AI060068Fatty acid binding protein30.001.25−0.16Metabolism
AA955115Carboxylesterase 3−0.321.17−0.30Metabolism
AI070183Nuclear protein 1−0.241.10−0.04Transcription
AA955423Fatty acid binding protein 5, epidermal−0.130.99NDMetabolism
AA859385Vimentin0.020.80−0.72Cytoskeleton
AA818904Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein−0.140.80−0.24Transcription
AA965232Serine (or cysteine) proteinase inhibitor, member 10.090.69−0.68Endopeptidase
AI044904Annexin VI0.080.61−0.79Calcium binding
AA964578Calpactin I heavy chain0.120.57−0.84Calcium binding
AI070507Fibromodulin0.030.51−0.63Extracellular matrix
AI059907Chemokine (C-X-C motif) ligand 10−0.140.47−0.11Signal transduction
AI059648Angiotensinogen−1.030.750.28Endopeptidase
AI060165Lipase, hormone sensitive−0.550.72NDMetabolism
AI1360483-hydroxy-3-methylglutaryl-Coenzyme A synthase 2−0.460.50−0.01Metabolism
AI136365Sortilin1−0.390.40−0.03Transport/Endocytosis
Cluster 2
AA901405Protein tyrosine phosphatase, non-receptor type 110.110.46−0.95Signal transduction
AA858569Neuronatin0.740.66−0.72Unclassified
AA963906RNA binding motif protein 160.510.02−0.64RNA binding
AA996833Aldolase C, fructose-biphosphate0.61−0.39−0.08Metabolism
AA900486ATP citrate lyase0.44−0.89−0.66Metabolism
AA818413Clusterin0.31−0.58NDApoptosis
Cluster 3
AA925370F-spondin0.04−0.970.79Extracellular matrix
AA859785Alcohol dehydrogenase 10.03−0.870.50Metabolism
AA859213Stearoyl-Coenzyme A desaturase 20.14−0.87−0.08Metabolism
AA819161Desmin−0.02−0.650.76Cytoskeleton
AA875489Voltage-dependent anion channel 10.26−0.76NDIon channel
Cluster 4
AA818559EST−0.880.090.03Unclassified
AA925361FK506-binding protein 1a−0.640.040.59Signal transduction
AA819832EST−0.590.000.86Unclassified
AA819638N-acetyltransferase 5−0.590.060.77Unclassified
AA859061EST−0.51−0.010.47Unclassified
AA859231Tropomyosin 1, alpha−0.46−0.181.32Cytoskeleton
AA818706Group specific component(vitamin D binding protein)−0.64−1.08−0.01Transport
AA924772Metallothionein 3−0.51−0.550.48Metal ion binding
AI058961Microtubule-associated protein 1b−0.680.050.61Cytoskeleton
AA875488EST−1.20−0.040.84Unclassified
AA819654Similar to zinc finger protein (LOC307362)−0.590.040.33Transcription
AA899387Similar to PD2 protein (LOC361531)−1.02−0.080.57Unclassified
AA955420Guanylate cyclase 1, soluble, beta 3−0.560.240.47Signal transduction
AA819832Period 1−0.590.000.86Signal transduction
Table 2

Genes differentially expressed in skeletal muscle of ZDF rats

Normalized expression (ZDF/ZLC)
Gene Name6W12W20WFunction
Expression ratios are shown by log2 scale of normalized mean signal intensities of ZDF rat genes to those of ZLC. W, weeks old.
Accession
 Cluster 1
AA819345Parvalbumin−0.02−1.180.86Calcium binding
AA818892Pyruvate dehydrogenase0.07−1.050.48Metabolism
AA963095Prohibitin−0.04−0.910.68Transcription/cell cycle
AA999182Solute carrier family 10, member 1−0.13−0.660.47Transport
AA817792EST−0.01−0.480.42Unclassified
AA924091Leprecan-like protein 2−0.02−0.310.83Oxidoreductase/metabolism
AA996896Ubiquitin-conjugating enzyme UBC70.39−0.08−0.12ubiquitin-conjugating
AA859043Triadin 10.36−0.650.25Translation/exocytosis
AA858950Regulator of G-protein signaling protein 20.91−0.250.05Signal transduction
AA924933Munc13-4 protein0.08−0.430.54Exocytosis
AA899619Alpha-globin transcription factor CP20.07−0.810.29Transcription
AI028932Prostaglandin F2 receptor negative regulator0.01−0.320.19Signal transduction
Cluster 2
AA900901Cyclin F−2.810.060.77Cell cycle
AA900958SorCS2−1.78−0.130.98Transport/Endocytosis
AA818691Myogenic factor 6−0.290.020.57Transcription
AA817748EST−1.11−0.101.29Unclassified
AA875328Fas-associated factor 1−0.79−0.030.79Apoptosis
AA957538Vesicular transport protein rvps45−0.60−0.170.54Transport
Cluster 3
AI136093Fatty acid amide hydrolase−0.810.120.07Metabolism
AA924224Cytochrome P450, subfamily 11B, polypeptide 2−0.490.24−0.02Biosynthesis
AA819300Squalene epoxidase−0.740.42−0.06Oxidoreductase/metabolism
AA818398Glutathione S-transferase, mu type 3 (Yb3)−0.490.35−0.08Transferase
AI072234p21 (CDKN1A)-activated kinase 1−0.490.290.05Signal transduction
AA859122EST−0.390.610.05Unclassified
AI136365Sortilin 1−0.390.40−0.03Transport/Endocytosis
AA900185EST−0.120.77−0.02Unclassified
AA925173Heparanase−0.110.63−0.30Extracellular matrix
Cluster 4
AA818605EST1.41−0.05−1.23Unclassified
AA859476Cofilin 10.76−0.28−0.18Cytoskeleton
AA819612Profilin0.58−0.16−0.06Cytoskeleton
AA859814Cystatin B0.57−0.06−0.24Structure
AA963445Selenoprotein P, plasma, 10.57−0.20−0.14Selenium binding
AA899180NAD(P)H dehydrogenase, quinone 10.87−1.990.02Oxydoreductase
AA900899Mitogen-activated protein kinase kinase 60.76−1.320.07Signal transduction
AA924727Collagen, type 1, alpha 11.28−1.04−0.05Extracellualr matrix
AA901017EST1.12−1.040.12Unclassified
AA962987Carbonyl reductase 10.97−0.76−0.05Oxydoreductase/metabolism
AA958018Cd24 antigen0.79−0.72−0.26Signal transduction
AA819336Cathepsin H0.65−0.32−0.01Endopeptidase
AA955881Fatty acid synthase0.32−0.130.05Metabolism
AA900486ATP citrate lyase0.44−0.89−0.89Metabolism
Cluster 5
AA899924Cd164 antigen0.181.01−0.31Signal transduction
AA924388WD repeat domain 20 isoform 1−0.171.00−0.84Meiosis
AA926010Fatty acid Coenzyme A ligase, long chain 20.250.770.77Metabolism
AA819897Synuclein, alpha0.210.25−0.51Signal transduction
AI070507Fibromodulin−0.100.32−0.50Extracellular matrix
Table 3

Genes differentially expressed in liver of ZDF rats

Normalized expression (ZDF/ZLC)
Gene Name6W12W20WFunction
Expression ratios are shown by log2 scale of normalized mean signal intensities of ZDF rat genes to those of ZLC. ND denotes “non-detectable or low signal-to-noise ratio”. W, weeks old.
Accession
 Cluster 1
AA859607Sterol-C4-methyl oxidase-like−0.04−0.651.58Metabolism
AA90093314-3-3 protein epsilon isoform−0.04−0.490.71Signal transduction
AA965012Zinc finger protein 354A−0.34−0.021.12Transcription
AA9248003-hydroxy-3-methylglutaryl-Coenzyme A synthase 1−0.270.001.36Metabolism
Cluster 2
AA859455Sir2-like 31.62−0.15−0.38Transcription
AA900766EST1.44−0.13−0.40Unclassified
AA998956Abl-interactor 21.240.12−0.49Signal transduction
AI059997Stress activated protein kinase alpha II0.96−0.02−0.04Signal transduction
AA818904Inhibitor of DNA binding 3, dominant negative helix-loop-helix protein0.940.01−0.17Transcription
AA955475Ubiquitin-conjugating enzyme E2D 20.88−0.22−0.19Ubiquitination
AA925792Superoxide dismutase 10.57−0.22−0.01Oxidoreductase
AA923836Eukaryotic initiation factor 5 (eIF-5)0.56−0.28−0.06Translation
AA964788Glutathione peroxidase 10.51−0.22−0.39Oxidoreductase
AA875555Protein kinase, mitogen activated 3 (ERK1)0.45−0.190.00Signal transduction
AA866249RAB4A, member RAS oncogene family0.92−1.670.03Signal transduction
AA818124Cytochrom P450 15-beta gene0.79−1.58−0.15Metabolism
AA818680Ornithine aminotransferase1.11−1.470.09Metabolism
AA818180Murine thymoma viral (v-akt) oncogene homolog 20.63−1.18−0.03Signal transduction
AA818043Cytochrome P450, 2c391.66−1.02−0.06Metabolism
AI145654Protein kinase, cAMP dependent regulatory, type I, alpha1.01−1.020.02Signal transduction
AA926256Neuritin0.92−0.97−0.02Axonogenesis
AA924591Cytochrome P450 4A30.87−0.940.00Metabolism
AA900486ATP citrate lyase0.44−0.89−0.66Metabolism
AA926359EST1.22−0.880.02Unclassified
AA925350Insulin-like growth factor binding protein, acid labile subunit0.55−0.840.02Signal transduction
AA901404EST1.82−0.83−0.36Unclassified
AA926010Fatty acid Coenzyme A ligase, long chain 21.20−0.780.04Metabolism
AA923919Cathepsin E1.09−0.67−0.19Endopeptidase
AA818406U6 snRNA-associated Sm-like protein LSm61.65−0.63−0.04RNA binding
AA875311Collagen, type V, alpha 20.92−0.60−0.04Extracellular matrix
AA859087Dynactin1.09−0.59−0.10Cytoskeleton
AA925794Diazepam binding inhibitor1.66−0.55−0.12Metabolism
AA819042Hemopexin0.91−0.50−0.04Endopeptidase
AA859963Cytochrome oxidase subunit VIc0.74−0.48−0.08Metabolism
AI136404Cytochrome P450 3A90.95−0.43−0.04Metabolism
AA859354G protein gamma-5 subunit0.60−0.40−0.03Signal transduction
AA818813Glutaredoxin 1 (thioltransferase)0.85−0.400.00Oxidoreductase
AA819611Insulin-like growth factor binding protein 31.08−0.37−0.08Signal transduction
AA955662Apolipoprotein C-I0.69−0.36−0.12Metabolism
AA819911Integrin beta 10.72−0.32−0.08Signal transduction
AA819465Apolipoprotein C-III0.85−0.32−0.12Metabolism
AA924594Cytochrome P450, 1a20.00−1.720.56Metabolism
Cluster 3
AA964628Glucose-6-phosphatase, catalytic0.080.81−0.50Metabolism
AI072234p21 (CDKN1A)-activated kinase 1−0.100.73−0.17Signal transduction
AI144577Regulator of G-protein signaling 12−0.160.700.00Signal transduction
AA964507Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein−0.081.10−0.24Transcription
AA924223Pyroglutamyl-peptidase I−0.340.940.00Proteolysis
AA926047Vacuolar protein sorting 35−0.330.69−0.36Transport/Endocytosis
AA924063Mitogen activated protein kinase kinase 1−0.400.58−0.04Signal transduction
AI059788Folate hydrolase−0.370.570.10Proteolysis
Cluster 4
AA859109EST−0.800.72−0.01Unclassified
AI145353Thyroid hormone receptor alpha−0.720.570.05Signal transduction
AA964978Phosphofructokinase, muscle−0.630.61−0.08Metabolism
AA819072Sgk−0.560.48NDSignal transduction
AA817985EST−0.550.63−0.02Unclassified
AA900733Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha−0.760.350.22Signal transduction
AA900759T-cell death associated gene−0.760.420.01Apoptosis
AA819663Acyl-CoA:dihydroxyacetonephosphate acyltransferase−0.930.010.08Metabolism
AA924572Kinesin family member 5B−1.120.60−0.11Cytoskeleton
AA901272EST−0.750.300.11Unclassified
AA924837Bone morphogenetic protein receptor, type 1A−0.660.390.11Signal transduction
AA819622Coagulation factor II receptor−1.190.220.03Signal transduction
AA925531Prolactin-like protein B−0.660.120.21Hormone
AA965187Lactalbumin, alpha−0.580.230.01Metabolism
Table 4

Concurrent expressed genes in adipose tissue, skeletal muscle and liver of ZDF rats

Tissue6W, NE12W, NE20W, NEDescription
Expression ratios are shown by log2 scale of normalized mean signal intensities of ZDF rat genes to those of ZLC. NE denotes “Normalized Expression(ZDF/ZLC)”. W, weeks old.
Accession
 Muscle & liver
AA875382muscle−0.170.47−0.41calbindin 1
liver1.07−0.640.02
AA819452muscle0.72−0.27−0.09transgelin
liver0.54−0.08−0.25
AA963225muscle0.66−0.16−0.07cathepsin B
liver0.47−0.16−0.04
AA963445muscle0.57−0.20−0.14selenoprotein P, plasma, 1
liver0.65−0.510.03
AA859385muscle0.68−0.63−0.23vimentin
liver0.87−0.20−0.07
AA924402muscle0.48−0.29−0.54general transcription factor IIa, 2
liver0.98−0.54−0.26
AA964960muscle0.87−1.39−0.26annexin 1
liver0.82−0.98−0.78
Muscle & fat
AA925443muscle−0.390.63−0.17cellular retinoic acid binding protein 2
fat−0.700.000.94
AA924564muscle−1.98−0.010.63mitochondrial ribosomal protein L11
fat−1.12−0.041.24
AA998065muscle−2.140.030.90collapsin response mediator protein 1
fat−1.72−0.070.61
AA866389muscle0.56−0.430.01lumican
fat0.53−0.08−0.53
AA924727muscle1.28−1.04−0.05collagen, type 1, alpha 1
fat0.71−0.54−0.65
AA997308muscle0.45−0.27−0.08enolase 2, gamma
fat0.45−0.35−0.24
AI043649muscle1.28−0.85−0.18growth associated protein 43
fat0.71−0.72−0.17
AI044424muscle0.71−1.16−0.23transgelin 3
fat0.68−0.62−0.28
Fat & liver
AA818443fat−0.01−0.250.65dipeptidylpeptidase 4
liver0.80−0.760.00
AI072330fat0.130.38−0.52lactate dehydrogenase A
liver1.31−0.05−0.38
AA925017fat0.050.65−0.58chemokine (C-X-C motif) ligand 11
liver0.72−0.55−0.01
AA956187fat0.060.60−1.05protein phosphatase V
liver0.67−0.430.00
AI145101fat−0.030.60−0.80hydroxyacyl glutathione hydrolase
liver0.70−0.830.00
Muscle & liver & fat
AA900053muscle0.97−0.32−0.14calpain, small subunit 1
fat0.300.41−1.24
liver1.11−0.63−0.32
AA925675muscle1.06−0.85−0.06collagen, type III, alpha 1
fat0.72−0.05−0.38
liver0.96−0.52−0.15
AA875221muscle0.98−0.07−1.07isocitrate dehydrogenase 3, gamma
fat−0.201.10−0.12
liver1.14−0.990.04
Figure 1
Figure 1

Hierachical Clustering Analysis of differentially expressed genes in insulin sensitive tissues. All replicated ratios of gene expression levels (shown with the Arabic numerals 1 to 4) are displayed colorimetically in the three ages (6-week, 6W; 12-week, 12W; 20-week, 20W) old of ZDF rats compared with the control. The scale bar at the bottom represents the relative level of expression of each gene. (Red) Increase and (green) decrease in expression level relative to the median value at specific ages of animals. Details of gene identity and expression differences are given in the Tables 1 to 3.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01679

Figure 2
Figure 2

Relative expression of select transcripts from the skeletal muscle and liver tissue of ZDF or ZLC rats by real-time RT-PCR. Total RNA was extracted from the skeletal muscle or liver of 6- and 12-week old rats, and PCR reactions were carried out in SYBR PCR core reagents. The y-axis is ΔΔ CT calculated after normalization to 18S rRNA or corresponding relative expression level of ZDF to ZLC rats under the microarray experiments. Grey bars, ΔΔCT; open bars, relative expression level (log2 scale) under the microarray experiments. Individual CT values are representative of means from triplicate measurements. Trdn, triadin 1; Pdh, pyruvate dehydrogenase; Map2k6, mitogen-activated protein kinase kinase 6, Pva, parvalbumin; Rgs2, regulator of G-protein signaling protein 2; Sort1, sortilin; Facl2, fatty acid coenzyme A ligase, long chain 2; Apoc1, apolipoprotein C-I; Mapk9, mitogen activated protein kinase 9; Mapk3, mitogen activated protein kinase 3; Rgs12, regulator of G-protein signaling protein 12; Capns, calpain, small subunit 1; Hpx, hemopexin.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01679

Figure 3
Figure 3

Quantitative expression of lipogenic genes in the liver or skeletal muscle at 6 weeks or 12 weeks of age. Total RNA was extracted from the 6- and 12-week old liver (A) or 6- and 12-week-old skeletal muscle (B) of ZDF and ZLC rats, and the PCR reactions were carried out in SYBR PCR core reagents. The y-axis is the relative expression level normalized against the mean expression level (2 ΔCT value) in 6-week old ZLC rats. Black bars, 6-week-old ZLC rats; open bars, 12-week-old ZLC rats; hatched bars, 6-week old ZDF rats; grey bars,12-week old ZDF rats. Liver and muscle tissues of two ZDF and ZLC rats were solubilized and Western blotted with PPARγ or SREBP1 or β-actin antibody (C). aP2, adipocyte fatty acid-binding protein; PPARγ, peroxisome proliferator activated receptor gamma; SREBP1c, sterol regulatory element-binding protein 1c; Facl2, fatty acid coenzyme A ligase, long chain 2; SCD1, stearoyl-CoA desaturase 1.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01679

Figure 4
Figure 4

Quantitative expression of antioxidant genes in the liver at 6 weeks and 12 weeks of age. Total RNA was extracted from the 6-week old liver or 12-week old liver of ZDF and ZLC rats, and the PCR reactions were carried out in SYBR PCR core reagents. The y-axis is the relative expression level normalized against the mean expression level (2ΔCT value) in 6-week old ZLC rats. Black bars, 6-week old ZLC rats; open bars, 12-week old ZLC rats; hatched bars, 6-week old ZDF rats; grey bars,12-week old ZDF rats.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01679

Figure 5
Figure 5

Quantitative expression of CYP genes in the liver at 6 weeks and 12 weeks of age. Total RNA was extracted from the 6-week or 12-week old liver of ZDF and ZLC rats, and PCR reactions were carried out in SYBR PCR core reagents. The y-axis is the relative expression level normalized against the mean expression level (2ΔCT value) in 6-week old ZLC rats. Black bars, 6-week-old ZLC rats; open bars, 12-week-old ZLC rats; hatched bars, 6-week-old ZDF rats; grey bars,12-week-old ZDF rats.

Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01679

We thank Dr Van-Anh Nguyen for critical review of the manuscript. This work was supported by the intramural grant of the National Institute of Health, Korea.

The array data have been submitted to the GEO data repository (http://www.ncbi.nlm.nih.gov/geo/) under accession no. GSE1080.

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  • Hermey G, Riedel IB, Hampe W, Schaller HC & Hermans-Borgmeyer I 1999 Identification and characterization of SorCS, a third member of a novel receptor family. Biochemical and Biophysical Research Communications 266 347–351.

    • Search Google Scholar
    • Export Citation
  • Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M & Hotamisligil GS 2002 A central role for JNK in obesity and insulin resistance. Nature 420 333–336.

    • Search Google Scholar
    • Export Citation
  • Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL & Bell GI 2000 Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nature Genetics 26 163–175.

    • Search Google Scholar
    • Export Citation
  • Imamura T, Vollenweider P, Egawa K, Clodi M, Ishibashi K, Nakashima N, Ugi S, Adams JW, Brown JH & Olefsky JM 1999 G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes. Molecular and Cellular Biology 19 6765–6774.

    • Search Google Scholar
    • Export Citation
  • Ishii M & Kurachi Y 2003 Physiological actions of regulators of G-protein signaling (RGS) proteins. Life Sciences 74 163–171.

  • Jones BH, Standridge MK & Moustaid N 1997 Angiotensin II increases lipogenesis in 3T3-L1 and human adipose cells. Endocrinology 138 1512–1519.

    • Search Google Scholar
    • Export Citation
  • Konrad D, Somwar R, Sweeney G, Yaworsky K, Hayashi M, Ramlal T & Klip A 2001 The antihyperglycemic drug alpha-lipoic acid stimulates glucose uptake via both GLUT4 translocation and GLUT4 activation: potential role of p38 mitogen-activated protein kinase in GLUT4 activation. Diabetes 50 1464–1471.

    • Search Google Scholar
    • Export Citation
  • Lam TK, Carpentier A, Lewis GF, van de Werve G, Fantus IG & Giacca A 2003 Mechanisms of the free fatty acid-induced increase in hepatic glucose production. American Journal of Physiology. Endocrinology and Metabolism 284 E863–E873.

    • Search Google Scholar
    • Export Citation
  • Lan H, Rabaglia ME, Stoehr JP, Nadler ST, Schueler KL, Zou F, Yandell BS & Attie AD 2003 Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility. Diabetes 52 688–700.

    • Search Google Scholar
    • Export Citation
  • Lewis GF, Carpentier A, Adeli K & Giacca A 2002 Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocrine Reviews 23 201–229.

    • Search Google Scholar
    • Export Citation
  • Lindgren CM, Nilsson A, Orho-Melander M, Almgren P & Groop LC 2001 Characterization of the annexin I gene and evaluation of its role in type 2 diabetes. Diabetes 50 2402–2405.

    • Search Google Scholar
    • Export Citation
  • Lopez-Alemany R, Redondo JM, Nagamine Y & Munoz-Canoves P 2003 Plasminogen activator inhibitor type-1 inhibits insulin signaling by competing with alphavbeta3 integrin for vitronectin binding. European Journal of Biochemistry 270 814–821.

    • Search Google Scholar
    • Export Citation
  • Luft FC 1997 G-proteins and insulin signaling. Journal of Molecular Medicine 75 233–235.

  • Malecki MT, Klupa T, Wanic K, Cyganek K, Frey J & Sieradzki J 2002 Vitamin D binding protein gene and genetic susceptibility to type 2 diabetes mellitus in a Polish population. Diabetes Research and Clinical Practice 57 99–104.

    • Search Google Scholar
    • Export Citation
  • Mazella J 2001 Sortilin/neurotensin receptor-3: a new tool to investigate neurotensin signaling and cellular trafficking? Cell Signalling 13 1–6.

    • Search Google Scholar
    • Export Citation
  • Melki V, Hullin F, Mazarguil H, Fauvel J, Ragab-Thomas JM & Chap H 1994 Annexin I as a potential inhibitor of insulin receptor protein tyrosine kinase. Biochemical and Biophysical Research Communications 203 813–819.

    • Search Google Scholar
    • Export Citation
  • Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D & Groop LC 2003 PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genetics 34 267–273.

    • Search Google Scholar
    • Export Citation
  • Morris NJ, Ross SA, Lane WS, Moestrup SK, Petersen CM, Keller SR & Lienhard GE 1998 Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes. Journal of Biological Chemistry 273 3582–3587.

    • Search Google Scholar
    • Export Citation
  • Nadler ST, Stoehr JP, Schueler KL, Tanimoto G, Yandell BS & Attie AD 2000 The expression of adipogenic genes is decreased in obesity and diabetes mellitus. PNAS 97 11371–11376.

    • Search Google Scholar
    • Export Citation
  • Pass GJ, Becker W, Kluge R, Linnartz K, Plum L, Giesen K & Joost HG 2002 Effect of hyperinsulinemia and type 2 diabetes-like hyperglycemia on expression of hepatic cytochrome p450 and glutathione s-transferase isoforms in a New Zealand obese-derived mouse backcross population. Journal of Pharmacology and Experimental Therapeutics 302 442–450.

    • Search Google Scholar
    • Export Citation
  • Petersen KF & Shulman GI 2002 Pathogenesis of skeletal muscle insulin resistance in type 2 diabetes mellitus. American Journal of Cardiology 90 11 G–18 G.

    • Search Google Scholar
    • Export Citation
  • Rocke DM & Durbin B 2001 A model for measurement error for gene expression arrays. Journal of Computational Biology 8 557–569.

  • Rome S, Clement K, Rabasa-Lhoret R, Loizon E, Poitou C, Barsh GS, Riou JP, Laville M & Vidal H 2003 Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp. Journal of Biological Chemistry 278 18063–18068.

    • Search Google Scholar
    • Export Citation
  • Saltiel AR 2001 New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell 104 517–529.

  • Schmitz-Peiffer C 2002 Protein kinase C and lipid-induced insulin resistance in skeletal muscle. Annals of the New York Academy of Sciences 967 146–157.

    • Search Google Scholar
    • Export Citation
  • Sreekumar R, Halvatsiotis P, Schimke JC & Nair KS 2002 Gene expression profile in skeletal muscle of type 2 diabetes and the effect of insulin treatment. Diabetes 51 1913–1920.

    • Search Google Scholar
    • Export Citation
  • Sreenan SK, Zhou YP, Otani K, Hansen PA, Currie KP, Pan CY, Lee JP, Ostrega DM, Pugh W, Horikawa Y, Cox NJ, Hanis CL, Burant CF, Fox AP, Bell GI & Polonsky KS 2001 Calpains play a role in insulin secretion and action. Diabetes 50 2013–2020.

    • Search Google Scholar
    • Export Citation
  • Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A & Klip A 1999 An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes. Journal of Biological Chemistry 274 10071–10078.

    • Search Google Scholar
    • Export Citation
  • Thummel KE & Schenkman JB 1990 Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat. Molecular Pharmacology 37 119–129.

    • Search Google Scholar
    • Export Citation
  • Yang X, Pratley RE, Tokraks S, Bogardus C & Permana PA 2002 Microarray profiling of skeletal muscle tissues from equally obese, non-diabetic insulin-sensitive and insulin-resistant Pima Indians. Diabetologia 45 1584–1593.

    • Search Google Scholar
    • Export Citation

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      Society for Endocrinology

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    Hierachical Clustering Analysis of differentially expressed genes in insulin sensitive tissues. All replicated ratios of gene expression levels (shown with the Arabic numerals 1 to 4) are displayed colorimetically in the three ages (6-week, 6W; 12-week, 12W; 20-week, 20W) old of ZDF rats compared with the control. The scale bar at the bottom represents the relative level of expression of each gene. (Red) Increase and (green) decrease in expression level relative to the median value at specific ages of animals. Details of gene identity and expression differences are given in the Tables 1 to 3.

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    Relative expression of select transcripts from the skeletal muscle and liver tissue of ZDF or ZLC rats by real-time RT-PCR. Total RNA was extracted from the skeletal muscle or liver of 6- and 12-week old rats, and PCR reactions were carried out in SYBR PCR core reagents. The y-axis is ΔΔ CT calculated after normalization to 18S rRNA or corresponding relative expression level of ZDF to ZLC rats under the microarray experiments. Grey bars, ΔΔCT; open bars, relative expression level (log2 scale) under the microarray experiments. Individual CT values are representative of means from triplicate measurements. Trdn, triadin 1; Pdh, pyruvate dehydrogenase; Map2k6, mitogen-activated protein kinase kinase 6, Pva, parvalbumin; Rgs2, regulator of G-protein signaling protein 2; Sort1, sortilin; Facl2, fatty acid coenzyme A ligase, long chain 2; Apoc1, apolipoprotein C-I; Mapk9, mitogen activated protein kinase 9; Mapk3, mitogen activated protein kinase 3; Rgs12, regulator of G-protein signaling protein 12; Capns, calpain, small subunit 1; Hpx, hemopexin.

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    Quantitative expression of lipogenic genes in the liver or skeletal muscle at 6 weeks or 12 weeks of age. Total RNA was extracted from the 6- and 12-week old liver (A) or 6- and 12-week-old skeletal muscle (B) of ZDF and ZLC rats, and the PCR reactions were carried out in SYBR PCR core reagents. The y-axis is the relative expression level normalized against the mean expression level (2 ΔCT value) in 6-week old ZLC rats. Black bars, 6-week-old ZLC rats; open bars, 12-week-old ZLC rats; hatched bars, 6-week old ZDF rats; grey bars,12-week old ZDF rats. Liver and muscle tissues of two ZDF and ZLC rats were solubilized and Western blotted with PPARγ or SREBP1 or β-actin antibody (C). aP2, adipocyte fatty acid-binding protein; PPARγ, peroxisome proliferator activated receptor gamma; SREBP1c, sterol regulatory element-binding protein 1c; Facl2, fatty acid coenzyme A ligase, long chain 2; SCD1, stearoyl-CoA desaturase 1.

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    Quantitative expression of antioxidant genes in the liver at 6 weeks and 12 weeks of age. Total RNA was extracted from the 6-week old liver or 12-week old liver of ZDF and ZLC rats, and the PCR reactions were carried out in SYBR PCR core reagents. The y-axis is the relative expression level normalized against the mean expression level (2ΔCT value) in 6-week old ZLC rats. Black bars, 6-week old ZLC rats; open bars, 12-week old ZLC rats; hatched bars, 6-week old ZDF rats; grey bars,12-week old ZDF rats.

  • View in gallery

    Quantitative expression of CYP genes in the liver at 6 weeks and 12 weeks of age. Total RNA was extracted from the 6-week or 12-week old liver of ZDF and ZLC rats, and PCR reactions were carried out in SYBR PCR core reagents. The y-axis is the relative expression level normalized against the mean expression level (2ΔCT value) in 6-week old ZLC rats. Black bars, 6-week-old ZLC rats; open bars, 12-week-old ZLC rats; hatched bars, 6-week-old ZDF rats; grey bars,12-week-old ZDF rats.

  • Baier LJ, Permana PA, Yang X, Pratley RE, Hanson RL, Shen GQ, Mott D, Knowler WC, Cox NJ, Horikawa Y, Oda N, Bell GI & Bogardus C 2000 A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. Journal of Clinical Investigation 106 R69–R73.

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    • Export Citation
  • Bastard JP, Pieroni L & Hainque B 2000 Relationship between plasma plasminogen activator inhibitor 1 and insulin resistance. Diabetes Metabolism Research and Reviews 16 192–201.

    • Search Google Scholar
    • Export Citation
  • Colombo C, Haluzik M, Cutson JJ, Dietz KR, Marcus-Samuels B, Vinson C, Gavrilova O & Reitman ML 2003 Opposite effects of background genotype on muscle and liver insulin sensitivity of lipoatrophic mice. Role of triglyceride clearance. Journal of Biological Chemistry 278 3992–3999.

    • Search Google Scholar
    • Export Citation
  • Diraison F, Dusserre E, Vidal H, Sothier M & Beylot M 2002 Increased hepatic lipogenesis but decreased expression of lipogenic gene in adipose tissue in human obesity. American Journal of Physiology. Endocrinology and Metabolism 282 E46–E51.

    • Search Google Scholar
    • Export Citation
  • Evans JL, Goldfine ID, Maddux BA & Grodsky GM 2002 Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocrine Reviews 23 599–622.

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    • Export Citation
  • Hainault I, Nebout G, Turban S, Ardouin B, Ferre P & Quignard-Boulange A 2002 Adipose tissue-specific increase in angiotensinogen expression and secretion in the obese (fa/fa) Zucker rat. American Journal of Physiology. Endocrinology and Metabolism 282 E59–E66.

    • Search Google Scholar
    • Export Citation
  • Hermey G, Riedel IB, Hampe W, Schaller HC & Hermans-Borgmeyer I 1999 Identification and characterization of SorCS, a third member of a novel receptor family. Biochemical and Biophysical Research Communications 266 347–351.

    • Search Google Scholar
    • Export Citation
  • Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M & Hotamisligil GS 2002 A central role for JNK in obesity and insulin resistance. Nature 420 333–336.

    • Search Google Scholar
    • Export Citation
  • Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL & Bell GI 2000 Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nature Genetics 26 163–175.

    • Search Google Scholar
    • Export Citation
  • Imamura T, Vollenweider P, Egawa K, Clodi M, Ishibashi K, Nakashima N, Ugi S, Adams JW, Brown JH & Olefsky JM 1999 G alpha-q/11 protein plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes. Molecular and Cellular Biology 19 6765–6774.

    • Search Google Scholar
    • Export Citation
  • Ishii M & Kurachi Y 2003 Physiological actions of regulators of G-protein signaling (RGS) proteins. Life Sciences 74 163–171.

  • Jones BH, Standridge MK & Moustaid N 1997 Angiotensin II increases lipogenesis in 3T3-L1 and human adipose cells. Endocrinology 138 1512–1519.

    • Search Google Scholar
    • Export Citation
  • Konrad D, Somwar R, Sweeney G, Yaworsky K, Hayashi M, Ramlal T & Klip A 2001 The antihyperglycemic drug alpha-lipoic acid stimulates glucose uptake via both GLUT4 translocation and GLUT4 activation: potential role of p38 mitogen-activated protein kinase in GLUT4 activation. Diabetes 50 1464–1471.

    • Search Google Scholar
    • Export Citation
  • Lam TK, Carpentier A, Lewis GF, van de Werve G, Fantus IG & Giacca A 2003 Mechanisms of the free fatty acid-induced increase in hepatic glucose production. American Journal of Physiology. Endocrinology and Metabolism 284 E863–E873.

    • Search Google Scholar
    • Export Citation
  • Lan H, Rabaglia ME, Stoehr JP, Nadler ST, Schueler KL, Zou F, Yandell BS & Attie AD 2003 Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility. Diabetes 52 688–700.

    • Search Google Scholar
    • Export Citation
  • Lewis GF, Carpentier A, Adeli K & Giacca A 2002 Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes. Endocrine Reviews 23 201–229.

    • Search Google Scholar
    • Export Citation
  • Lindgren CM, Nilsson A, Orho-Melander M, Almgren P & Groop LC 2001 Characterization of the annexin I gene and evaluation of its role in type 2 diabetes. Diabetes 50 2402–2405.

    • Search Google Scholar
    • Export Citation
  • Lopez-Alemany R, Redondo JM, Nagamine Y & Munoz-Canoves P 2003 Plasminogen activator inhibitor type-1 inhibits insulin signaling by competing with alphavbeta3 integrin for vitronectin binding. European Journal of Biochemistry 270 814–821.

    • Search Google Scholar
    • Export Citation
  • Luft FC 1997 G-proteins and insulin signaling. Journal of Molecular Medicine 75 233–235.

  • Malecki MT, Klupa T, Wanic K, Cyganek K, Frey J & Sieradzki J 2002 Vitamin D binding protein gene and genetic susceptibility to type 2 diabetes mellitus in a Polish population. Diabetes Research and Clinical Practice 57 99–104.

    • Search Google Scholar
    • Export Citation
  • Mazella J 2001 Sortilin/neurotensin receptor-3: a new tool to investigate neurotensin signaling and cellular trafficking? Cell Signalling 13 1–6.

    • Search Google Scholar
    • Export Citation
  • Melki V, Hullin F, Mazarguil H, Fauvel J, Ragab-Thomas JM & Chap H 1994 Annexin I as a potential inhibitor of insulin receptor protein tyrosine kinase. Biochemical and Biophysical Research Communications 203 813–819.

    • Search Google Scholar
    • Export Citation
  • Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E, Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P, Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D & Groop LC 2003 PGC-1 alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genetics 34 267–273.

    • Search Google Scholar
    • Export Citation
  • Morris NJ, Ross SA, Lane WS, Moestrup SK, Petersen CM, Keller SR & Lienhard GE 1998 Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes. Journal of Biological Chemistry 273 3582–3587.

    • Search Google Scholar
    • Export Citation
  • Nadler ST, Stoehr JP, Schueler KL, Tanimoto G, Yandell BS & Attie AD 2000 The expression of adipogenic genes is decreased in obesity and diabetes mellitus. PNAS 97 11371–11376.

    • Search Google Scholar
    • Export Citation
  • Pass GJ, Becker W, Kluge R, Linnartz K, Plum L, Giesen K & Joost HG 2002 Effect of hyperinsulinemia and type 2 diabetes-like hyperglycemia on expression of hepatic cytochrome p450 and glutathione s-transferase isoforms in a New Zealand obese-derived mouse backcross population. Journal of Pharmacology and Experimental Therapeutics 302 442–450.

    • Search Google Scholar
    • Export Citation
  • Petersen KF & Shulman GI 2002 Pathogenesis of skeletal muscle insulin resistance in type 2 diabetes mellitus. American Journal of Cardiology 90 11 G–18 G.

    • Search Google Scholar
    • Export Citation
  • Rocke DM & Durbin B 2001 A model for measurement error for gene expression arrays. Journal of Computational Biology 8 557–569.

  • Rome S, Clement K, Rabasa-Lhoret R, Loizon E, Poitou C, Barsh GS, Riou JP, Laville M & Vidal H 2003 Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp. Journal of Biological Chemistry 278 18063–18068.

    • Search Google Scholar
    • Export Citation
  • Saltiel AR 2001 New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell 104 517–529.

  • Schmitz-Peiffer C 2002 Protein kinase C and lipid-induced insulin resistance in skeletal muscle. Annals of the New York Academy of Sciences 967 146–157.

    • Search Google Scholar
    • Export Citation
  • Sreekumar R, Halvatsiotis P, Schimke JC & Nair KS 2002 Gene expression profile in skeletal muscle of type 2 diabetes and the effect of insulin treatment. Diabetes 51 1913–1920.

    • Search Google Scholar
    • Export Citation
  • Sreenan SK, Zhou YP, Otani K, Hansen PA, Currie KP, Pan CY, Lee JP, Ostrega DM, Pugh W, Horikawa Y, Cox NJ, Hanis CL, Burant CF, Fox AP, Bell GI & Polonsky KS 2001 Calpains play a role in insulin secretion and action. Diabetes 50 2013–2020.

    • Search Google Scholar
    • Export Citation
  • Sweeney G, Somwar R, Ramlal T, Volchuk A, Ueyama A & Klip A 1999 An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes. Journal of Biological Chemistry 274 10071–10078.

    • Search Google Scholar
    • Export Citation
  • Thummel KE & Schenkman JB 1990 Effects of testosterone and growth hormone treatment on hepatic microsomal P450 expression in the diabetic rat. Molecular Pharmacology 37 119–129.

    • Search Google Scholar
    • Export Citation
  • Yang X, Pratley RE, Tokraks S, Bogardus C & Permana PA 2002 Microarray profiling of skeletal muscle tissues from equally obese, non-diabetic insulin-sensitive and insulin-resistant Pima Indians. Diabetologia 45 1584–1593.

    • Search Google Scholar
    • Export Citation