Mitochondrial gene expression and increased oxidative metabolism: role in increased lifespan of fat-specific insulin receptor knock-out mice

Caloric restriction, leanness and decreased activity of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling are associated with increased longevity in a wide range of organisms from Caenorhabditis elegans to humans. Fat-specific insulin receptor knock-out (FIRKO) mice represent an interesting dichotomy, with leanness and increased lifespan, despite normal or increased food intake. To determine the mechanisms by which a lack of insulin signaling in adipose tissue might exert this effect, we performed physiological and gene expression studies in FIRKO and control mice as they aged. At the whole body level, FIRKO mice demonstrated an increase in basal metabolic rate and respiratory exchange ratio. Analysis of gene expression in white adipose tissue (WAT) of FIRKO mice from 6 to 36 months of age revealed persistently high expression of the nuclear-encoded mitochondrial genes involved in glycolysis, tricarboxylic acid cycle, β-oxidation and oxidative phosphorylation as compared to expression of the same genes in WAT from controls that showed a tendency to decline in expression with age. These changes in gene expression were correlated with increased cytochrome c and cytochrome c oxidase subunit IV at the protein level, increased citrate synthase activity, increased expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and PGC-1β, and an increase in mitochondrial DNA in WAT of FIRKO mice. Together, these data suggest that maintenance of mitochondrial activity and metabolic rates in adipose tissue may be important contributors to the increased lifespan of the FIRKO mouse.     

Reconstitution of insulin action in muscle, white adipose tissue, and brain of insulin receptor knock-out mice fails to rescue diabetes

Type 2 diabetes results from an impairment of insulin action. The first demonstrable abnormality of insulin signaling is a decrease of insulin-dependent glucose disposal followed by an increase in hepatic glucose production. In an attempt to dissect the relative importance of these two changes in disease progression, we have employed genetic knock-outs/knock-ins of the insulin receptor. Previously, we demonstrated that insulin receptor knock-out mice (Insr(-/-)) could be rescued from diabetes by reconstitution of insulin signaling in liver, brain, and pancreatic β cells (L1 mice). In this study, we used a similar approach to reconstitute insulin signaling in tissues that display insulin-dependent glucose uptake. Using GLUT4-Cre mice, we restored InsR expression in muscle, fat, and brain of Insr(-/-) mice (GIRKI (Glut4-insulin receptor knock-in line 1) mice). Unlike L1 mice, GIRKI mice failed to thrive and developed diabetes, although their survival was modestly extended when compared with Insr(-/-). The data underscore the role of developmental factors in the presentation of murine diabetes. The broader implication of our findings is that diabetes treatment should not necessarily target the same tissues that are responsible for disease pathogenesis.     

Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue

Several lines of evidence suggest that a functional relationship exists between caveolin-1 and insulin signaling. However, it remains unknown whether caveolin-1 is normally required for proper insulin receptor signaling in vivo. To address this issue, we examined the status of insulin receptor signaling in caveolin-1 (–/–)-deficient (Cav-1 null) mice. Here, we show that Cav-1 null mice placed on a high-fat diet for 9 mo develop postprandial hyperinsulinemia. An insulin tolerance test (ITT) revealed that young Cav-1 null mice on a normal chow diet are significantly unresponsive to insulin, compared with their wild-type counterparts. This insulin resistance is due to a primary defect in adipose tissue, as evidenced by drastically reduced insulin receptor protein levels (>90%), without any changes in insulin receptor mRNA levels. These data suggest that caveolin-1 acts as a molecular chaperone that is necessary for the proper stabilization of the insulin receptor in adipocytes in vivo. In support of this notion, we demonstrate that recombinant expression of caveolin-1 in Cav-1 null mouse embryo fibroblasts rescues insulin receptor protein expression. These data provide evidence that the lean body phenotype observed in the Cav-1 knockout mice is due, at least in part, to a defect in insulin-regulated lipogenesis. caveolae; caveolin; insulin signaling; protein stabilization; knockout mice     

Weight loss in tumour-bearing mice is not associated with changes in resistin gene expression in white adipose tissue.

Resistin, a product of white adipose tissue, is postulated to induce insulin resistance in obesity and regulate adipocyte differentiation. The aim of this study was to examine resistin gene expression in adipose tissue from mice bearing the MAC16 adenocarcinoma, which induces cancer cachexia with marked wasting of adipose tissue and skeletal muscle mass. MAC16-bearing mice lost weight progressively over the period following tumour transplantation, while the weight of control mice remained stable. Leptin mRNA in gonadal fat was 50 % lower in MAC16 mice than in controls (p < 0.05). Plasma insulin concentrations were also significantly lower in the MAC16 group (p < 0.05). However, resistin mRNA level in gonadal fat in MAC16 mice was similar to controls (94 % of controls). Thus, despite severe weight loss and significant falls in leptin expression and insulin concentration, resistin gene expression appears unchanged in white adipose tissue of mice with MAC16 tumour. Maintenance of resistin production may help inhibit the formation of new adipocytes in cancer cachexia.     

Reduced expression of PGC-1 and insulin-signaling molecules in adipose tissue is associated with insulin resistance

Peroxisome proliferator-activated receptor gamma (PPAR gamma) co-activator 1 (PGC-1) regulates glucose metabolism and energy expenditure and, thus, potentially insulin sensitivity. We examined the expression of PGC-1, PPAR gamma, insulin receptor substrate-1 (IRS-1), glucose transporter isoform-4 (GLUT-4), and mitochondrial uncoupling protein-1 (UCP-1) in adipose tissue and skeletal muscle from non-obese, non-diabetic insulin-resistant, and insulin-sensitive individuals. PGC-1, both mRNA and protein, was expressed in human adipose tissue and the expression was significantly reduced in insulin-resistant subjects. The expression of PGC-1 correlated with the mRNA levels of IRS-1, GLUT-4, and UCP-1 in adipose tissue. Furthermore, the adipose tissue expression of PGC-1 and IRS-1 correlated with insulin action in vivo. In contrast, no differential expression of PGC-1, GLUT-4, or IRS-1 was found in the skeletal muscle of insulin-resistant vs insulin-sensitive subjects. The findings suggest that PGC-1 may be involved in the differential gene expression and regulation between adipose tissue and skeletal muscle. The combined reduction of PGC-1 and insulin signaling molecules in adipose tissue implicates adipose tissue dysfunction which, in turn, can impair the systemic insulin response in the insulin-resistant subjects.     

Zinc-alpha 2-glycoprotein gene expression in adipose tissue is related with insulin resistance and lipolytic genes in morbidly obese patients

Objective

Zinc-α₂ glycoprotein (ZAG) stimulates lipid loss by adipocytes and may be involved in the regulation of adipose tissue metabolism. However, to date no studies have been made in the most extreme of obesity. The aims of this study are to analyze ZAG expression levels in adipose tissue from morbidly obese patients, and their relationship with lipogenic and lipolytic genes and with insulin resistance (IR).

Methods

mRNA expression levels of PPARγ, IRS-1, IRS-2, lipogenic and lipolytic genes and ZAG were quantified in visceral (VAT) and subcutaneous adipose tissue (SAT) of 25 nondiabetic morbidly obese patients, 11 with low IR and 14 with high IR. Plasma ZAG was also analyzed.

Results

The morbidly obese patients with low IR had a higher VAT ZAG expression as compared with the patients with high IR (p = 0.023). In the patients with low IR, the VAT ZAG expression was greater than that in SAT (p = 0.009). ZAG expression correlated between SAT and VAT (r = 0.709, p<0.001). VAT ZAG expression was mainly predicted by insulin, HOMA-IR, plasma adiponectin and expression of adiponectin and ACSS2. SAT ZAG expression was only predicted by expression of ATGL.

Conclusions

ZAG could be involved in modulating lipid metabolism in adipose tissue and is associated with insulin resistance. These findings suggest that ZAG may be a useful target in obesity and related disorders, such as diabetes.     

Resistin and RELM-alpha gene expression in white adipose tissue of lactating mice

An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.     

Cloning, expression, and mapping of a gene that is upregulated in adipose tissue of mice deficient in bombesin receptor subtype-3.

To identify novel obesity-related genes in adipose tissue, differential display was performed using bombesin receptor subtype-3 (BRS-3)-deficient mice. These mice exhibit mild late-onset obesity. We report that a gene, Urb, is upregulated in these mice. Full-length Urb cDNA is approximately 3 kb long and comprises an open reading frame of 949 amino acid residues. Interestingly, Urb mRNA expression in brown adipose tissue of BRS-3-deficient mice is fourfold higher than that in wild-type controls. Enhanced Urb mRNA expression was also observed in brain, digestive tissues, kidney, and lung. Within the brain, Urb mRNA is detected in the dorsal endopiriform nucleus and choroid plexus. A T31 radiation hybrid mapping panel revealed that the Urb gene maps to mouse chromosome 16. Collectively, these findings suggest that Urb may have a unique function in the regulation of body weight and energy metabolism.     

Extension of coronary artery disease is associated with increased IL-6 and decreased adiponectin gene expression in epicardial adipose tissue

Epicardial adipose tissue (EAT) expresses lower levels of adiponectin in patients with CAD and higher levels of inflammatory mediators such as IL-6 and leptin than subcutaneous adipose tissue. This showed one important role of EAT in coronary artery disease. However, the relationship of EAT adiponectin and IL-6 levels to the extension of coronary artery disease has not hitherto been determined. We sought to determine whether the levels of adiponectin and interleukin-6 (IL-6) mRNA in epicardial adipose tissue are associated with the extension of coronary artery disease (CAD). Methods: Angiographic and hormones expression were evaluated from epicardial and subcutaneous adipose tissue. 92 patients (58 CAD, 34 non-CAD) who underwent cardiac surgery. Adiponectin and IL-6 mRNA levels were measured by real time RT-PCR in epicardial and subcutaneous adipose tissue (SAT) following angiographic evaluation of their coronary arteries. Results: We found that epicardial adipose tissue of CAD expressed lower levels of adiponectin mRNA and higher levels of IL-6 mRNA than that of non-CAD patients. As the number of injured arteries rose, adiponectin mRNA levels decreased (r = −0.402, p < 0.001) and IL-6 mRNA increased (r = 0.514, p < 0.001) in epicardial adipose tissue. Conclusions: The extension of CAD is significantly associated with the expression of adiponectin and IL-6 mRNA in EAT. These findings suggest that low adiponectin and high IL-6 expression by EAT may contribute to CAD extension.     

Intrinsic differences of insulin receptor kinase activity in red and white muscle

The sensitivity and responsiveness of glucose uptake and glycogen synthesis to insulin are 3-4-fold greater in red than in white skeletal muscle (James, D. E., Jenkins, A. B., and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574). In the present study, the insulin receptor tyrosine kinase activity has been examined in red and white muscle of rats. Partially purified insulin receptors were obtained from muscle following solubilization in detergent, ultracentrifugation, and lectin affinity chromatography. Total insulin receptor number per gram of tissue was slightly higher in red (30%) than in white muscle. In contrast, basal and insulin-stimulated autophosphorylation, normalized for receptor number, were 2.3-fold higher in red muscle. A similar difference was observed in the ability of partially purified receptors to phosphorylate the exogenous substrate polyglutamate/tyrosine. The integrity of the insulin receptor preparation in the two fiber types was identical as determined by affinity cross-linking of [125I-TyrB26]insulin to the receptor. Mixing partially purified receptors from red and white muscle resulted in an additive response for exogenous substrate phosphorylation, suggesting that the difference in tyrosine kinase activity was not due to the presence of an inhibitor or activator. The results suggest that there are differences in the insulin receptors of red and white muscles that lead to discordance in their basal and insulin-stimulated intrinsic tyrosine kinase activity. The correlation between these differences and insulin action in red and white muscle supports the concept that the insulin receptor tyrosine kinase activity is involved in the initiation of insulin action.     

MicroRNA expression in abdominal and gluteal adipose tissue is associated with mRNA expression levels and partly genetically driven

To understand how miRNAs contribute to the molecular phenotype of adipose tissues and related traits, we performed global miRNA expression profiling in subcutaneous abdominal and gluteal adipose tissue of 70 human subjects and characterised which miRNAs were differentially expressed between these tissues. We found that 12% of the miRNAs were significantly differentially expressed between abdominal and gluteal adipose tissue (FDR adjusted p<0.05) in the primary study, of which 59 replicated in a follow-up study of 40 additional subjects. Further, 14 miRNAs were found to be associated with metabolic syndrome case-control status in abdominal tissue and three of these replicated (primary study: FDR adjusted p<0.05, replication: p<0.05 and directionally consistent effect). Genome-wide genotyping was performed in the 70 subjects to enable miRNA expression quantitative trait loci (eQTL) analysis. Candidate miRNA eQTLs were followed-up in the additional 40 subjects and six significant, independent cis-located miRNA eQTLs (primary study: p<0.001; replication: p<0.05 and directionally consistent effect) were identified. Finally, global mRNA expression profiling was performed in both tissues to enable association analysis between miRNA and target mRNA expression levels. We find 22% miRNAs in abdominal and 9% miRNAs in gluteal adipose tissue with expression levels significantly associated with the expression of corresponding target mRNAs (FDR adjusted p<0.05). Taken together, our results indicate a clear difference in the miRNA molecular phenotypic profile of abdominal and gluteal adipose tissue, that the expressions of some miRNAs are influenced by cis-located genetic variants and that miRNAs are associated with expression levels of their predicted mRNA targets.     

The ratio of estrogen receptor alpha to estrogen receptor beta in adipose tissue is associated with leptin production and obesity

The loss of estrogen associated with menopause is suspected to play an important regulatory role in changes of fat metabolism and obesity. To evaluate the relationship between obesity and the ratio of estrogen receptor subtypes (ERalpha/ERbeta) in adipose tissues in pre- and postmenopausal women, we measured the anthropometric indices of 31 premenopausal women and 12 postmenopausal women. Serum samples, subcutaneous and omental adipose tissues were also obtained from study participants. Serum leptin, adiponectin, IL-6, and TNF-alpha levels were measured using ELISA methods. Real-time RT-PCR analysis was performed to detect and to compare mRNA levels of leptin and estrogen receptor subtypes (ERalpha and ERbeta) from adipose tissues. The ratio of abdominal subcutaneous to omental adipose tissue for the ER subtypes (Sc-Om ratio of the ER subtypes), i.e., subcutaneous ERalpha/ERbeta over omental ERalpha/ERbeta, showed significant correlations with anthropometric indices including BMI (r=0.801, p<0.05) and waist circumference (r=0.696, p<0.05) in both pre- and postmenopausal women. The Sc-Om ratio of the ER subtypes also had a significant correlation with the serum leptin level (r=0.735, p<0.05) as well as the mRNA level of leptin in omental adipose tissue (r=0.753, p<0.05). However, there were no significant differences between the pre- and postmenopausal groups with regard to the expressed level of ER subtypes. In conclusion, our study results showed that the ratio of ERalpha to ERbeta in adipose tissue was associated with obesity as well as the serum level and production of leptin in omental adipose tissue.     

Dissociation between adipose tissue fluxes and lipogenic gene expression in ob/ob mice

Recent evidence has been presented that expression of lipogenic genes is downregulated in adipose tissue of ob/ob mice as well as in human obesity, suggesting a functionally lipoatrophic state. Using (2)H(2)O labeling, we measured three adipose tissue biosynthetic processes concurrently: triglyceride (TG) synthesis, palmitate de novo lipogenesis (DNL), and cell proliferation (adipogenesis). To determine the effect of the ob/ob mutation (leptin deficiency) on these parameters, adipose dynamics were compared in ob/ob, leptin-treated ob/ob, food-restricted ob/ob, and lean control mice. Adipose tissue fluxes for TG synthesis, de novo lipogenesis (DNL), and adipogenesis were dramatically increased in ob/ob mice compared with lean controls. Low-dose leptin treatment (2 microg/day) via miniosmotic pump suppressed all fluxes to control levels or below. Food restriction in ob/ob mice only modestly reduced DNL, with no change in TG synthesis or adipogenesis. Measurement of mRNA levels in age-matched ob/ob mice showed generally normal expression levels for most of the selected lipid anabolic genes, and leptin treatment had, with few exceptions, only modest effects on their expression. We conclude that leptin deficiency per se results in marked elevations in flux through diverse lipid anabolic pathways in adipose tissue (DNL, TG synthesis, and cell proliferation), independent of food intake, but that gene expression fails to reflect these changes in flux.     

Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu

Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17beta-estradiol (17beta-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17beta-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17beta-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER)alpha and ERbeta densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ERalpha predominated over ERbeta expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17beta-E2 increased ERalpha (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.     

Pulmonary hypertension in TNF-alpha-overexpressing mice is associated with decreased VEGF gene expression.

Tumor necrosis factor-alpha (TNF-alpha) transgenic mice have previously been found to have characteristics consistent with emphysema and severe pulmonary hypertension. Lungs demonstrated alveolar enlargement as well as interstitial thickening due to chronic inflammation and perivascular fibrosis. In the present report, we sought to determine potential mechanisms leading to development of pulmonary hypertension in TNF-alpha transgenic mice. To determine whether sustained vasoconstriction was an important component of this pulmonary hypertension, nitric oxide was administered and hemodynamics were measured. Nitric oxide (25 ppm) failed to normalize right ventricular pressure in transgene-positive mice, suggesting that the pulmonary hypertension was not due to sustained vasoconstriction. Structural analysis of the pulmonary arteries found adventitial thickening and a trend toward medial hypertrophy in pulmonary arteries of transgene-positive mice, suggesting that vascular remodeling had occurred. Echocardiographic measurement of the percent fractional shortening of the left ventricle as a measurement of ventricular function in vivo revealed that left ventricular dysfunction was not contributing to pulmonary hypertension. We examined expression of genes known to be important in regulation of vascular tone and structure. Messenger RNA expression of vascular endothelial growth factor and its receptor flk-1 was reduced compared with transgene-negative littermates at all ages. Endothelial and inducible nitric oxide synthase mRNA levels were similar in both groups. Endothelin-1 mRNA was also decreased in TNF-alpha transgenic mice. Interestingly, female transgenic mice had decreased survival rate compared with male transgenic mice. We conclude that chronic overexpression of TNF-alpha is associated with decreased vascular endothelial growth factor and flk-1 gene expression, pulmonary vascular remodeling, and severe pulmonary hypertension, although the precise mechanism is unknown.