Differential response of obese gene expression from fasting in bovine adipose tissues

To understand the molecular mechanism for intramuscular fat deposition, the expression of the obese gene was examined in response to fasting. Food deprivation for 48 h induced a decrease in the level of obese mRNA in pooled adipose tissues (abdominal, perirenal, subcutaneous, intermuscular and intramuscular). The expression of obese mRNA was examined for individual adipose tissue from several fat depots. It was highly expressed in perirenal adipose tissue, but fasting did not affect its expression level in this tissue. Moderate levels were detected in subcutaneous and intermuscular adipose tissues, and a fasting-induced decrease in obese mRNA was apparent in these tissues. The expression level of the obese gene in intramuscular adipose tissue was very low and did not respond to fasting.     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa).

To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa). In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues. Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold). The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues. There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats. LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold). The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold). Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity.     

The age-related differences in obese and fatty acid synthase gene expression in white adipose tissue of rat

To determine if the age-dependent increase of adiposity is directly related to altered obese (ob) and fatty acid synthase (FAS) gene expression, we assessed an adiposity index, leptin and FAS mRNA levels, FAS activity in perirenal adipose tissue and serum leptin concentration in rats aged 1, 2, 3, 6 and 20 months. The results indicate that there are two distinct phases of changes in perirenal white adipose tissue leptin mRNA level and serum leptin concentration. The first phase, between 1 and 3 months of the animals' lives, was characterized by a strong positive correlation between adiposity index and leptin mRNA level as well as serum leptin concentration. In the second phase (over 3 months) no significant changes of leptin mRNA and serum concentration occurred. A close correlation between the age-induced increase of leptin mRNA abundance and serum leptin concentration and the age-induced suppression of FAS gene expression in the same tissue was observed. This suggests that the changes of FAS gene expression occur in response to serum leptin concentration and that in mature rats the high level of ob gene expression and consequently the high leptin concentration protect the white adipose tissue cells against fat overload by two independent mechanisms: (a) preventing an increase of food intake through the leptin action on the hypothalamus; (b) inhibiting FAS gene expression and consequently decreasing the rate of lipogenesis.     

Metallothionein gene expression in human adipose tissue from lean and obese subjects.

Expression of the gene encoding metallothionein, a low molecular-weight cysteine-rich, stress-response and metal-binding protein was examined in human adipose tissue. The mRNA for MT-2A, a major metallothionein isoform in humans, was detected in subcutaneous fat using a specific antisense oligonucleotide probe. The level of MT-2A mRNA was significantly higher in a group of obese subjects than in a lean group, paralleling a similar increase in ob mRNA. A two-week period on a diet of 800 calories/day did not lead to any significant change in MT-2 mRNA levels. Separation of mature adipocytes from the cells of the stromal vascular fraction indicated that in human adipose tissue the metallothionein (MT-2A) gene is expressed both in adipocytes and in other cells of the tissue.     

Perilipin Expression in Human Adipose Tissues: Effects of Severe Obesity,Gender, and Depot

Objective: Perilipins are phosphoproteins that are localized to the surface of triacylglycerol droplets within adipocytes where they regulate the rate of lipolysis. We sought to determine the effects of severe obesity and depot [omental (Om) vs. subcutaneous (Sc)] on perilipin expression in the adipose tissue of individuals. Research Methods and Procedures: Samples of Om and Sc adipose tissues obtained at surgery from severely obese subjects and fat aspirations from nonobese subjects were analyzed for perilipin protein and mRNA levels by Northern and Western analysis. Results: Perilipin A (periA) was the major perilipin expressed in adipose tissues. periA mRNA relative abundance was significantly lower in Sc adipose tissue from severely obese compared to that from nonobese subjects. Western blotting of adipose tissue extracts showed that periA protein levels expressed relative to tissue protein or fat cell surface area were significantly lower (～ ?40%) in abdominal Sc adipose tissue from severely obese compared to that from nonobese subjects. However, the calculated mass of perilipin per fat cell did not differ between the two groups. Perilipin mRNA levels were higher in Sc compared to Om adipose tissue from obese individuals (p < 0.025; n = 26; 17 women, 9 men); however, periA protein levels did not differ. In addition, perilipin protein, but not mRNA, levels were higher in Sc adipose tissue from obese men than from women (p < 0.025). Discussion: Variations in perilipin expression may contribute to the higher basal lipolytic rates observed in obese compared to nonobese individuals and in obese women compared to obese men.     

Regulation of haptoglobin gene expression in 3T3-L1 adipocytes by cytokines, catecholamines, and PPARgamma

Factors which regulate expression of the haptoglobin (acute phase reactant) gene in adipocytes have been examined using 3T3-L1 cells. Haptoglobin expression was observed by Northern blotting in each of the major white adipose tissue depots of mice (epididymal, subcutaneous, mesenteric, and perirenal) and in interscapular brown fat. Expression occurred in mature adipocytes, but not in the stromal-vascular fraction. In 3T3-L1 cells, haptoglobin mRNA was detected from day 4 after the induction of differentiation into adipocytes. Lipopolysaccharide and the cytokines, TNFalpha and interleukin-6, resulted in substantial increases in haptoglobin mRNA in 3T3-L1 adipocytes; the increase (7-fold) was highest with TNFalpha. Increases in haptoglobin mRNA level were also induced by dexamethasone, noradrenaline, isoprenaline, and a beta3-adrenoceptor agonist. In contrast, haptoglobin mRNA was reduced by nicotinic acid and the PPARgamma agonist, rosiglitazone. RT-PCR showed that the haptoglobin gene was expressed in human adipose tissue (subcutaneous, omental). It is concluded that haptoglobin gene expression in adipocytes is stimulated by inflammatory cytokines, glucocorticoids, and the sympathetic system, while activation of the PPARgamma nuclear receptor is strongly inhibitory.     

Metallothionein gene expression and secretion in white adipose tissue

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese (ob/ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a beta(3)-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.     

Induction of uncoupling protein-2 (UCP2) gene expression on the differentiation of rat preadipocytes to adipocytes in primary culture

We have examined uncoupling protein-2 (UCP2) gene expression in the adipose tissue of obese and normal rats and mice, and also in differentiated rat adipocytes in primary culture. Expression of the UCP2 gene was examined in rat and mouse adipose tissues using both RT-PCR and Northern blotting. Although the RT-PCR was not quantitative, the band corresponding to the UCP2 mRNA was stronger in white adipose tissue than in brown fat, regardless of the body weight of the rats. In agreement with the RT-PCR data, there was a higher level of UCP2 mRNA in the white adipocytes than in brown adipocytes, the level being greater in obese mice. Fibroblastic preadipocytes were obtained from the inguinal fat pad of suckling rats. Lipid droplets developed inside the cells upon differentiation and adipsin and UCP2 mRNAs were detected by Northern blotting. Both mRNAs were evident in the adipocytes at 4, 6, and 10 d after the induction of differentiation. There was no indication that the expression of UCP2 was markedly affected by the addition of leptin, dexamethasone or isoprenaline.     

Unusual increase of Scd1 and Elovl6 expression in rat inguinal adipose tissue

It is generally accepted that the location of body fat deposits may play an important role in the risk of developing some endocrine and metabolic diseases. We have studied the effect of food restriction and food restriction/refeeding, often practiced by individuals trying to lose body weight, on the expression of genes which are associated with obesity and certain metabolic disorders in inguinal, epididymal, and perirenal rat white adipose tissues. Gene expression was analyzed by real time semi-quantitative polymerase chain reaction and by Western blot. We found that prolonged food restriction caused a significant decrease of body and adipose tissue mass as well as the increase of Scd1 and Elovl6 gene expressions in all main rat adipose tissue deposits. Food restriction/refeeding caused increases of: a) Scd1 and Elovl6 mRNA levels in adipose tissue, b) Scd1 protein level and c) desaturation index in adipose tissue. The increased expression of both genes was unusually high in inguinal adipose tissue. The results suggest that the increase of Scd1 and Elovl6 gene expressions in white adipose tissue by prolonged food restriction and prolonged food restriction/refeeding may contribute to accelerated fat recovery that often occurs in individuals after food restriction/refeeding.     

A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor gamma. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function.     

Diversity of SREBP-1 gene expression in rat adipose tissue depots in response to refeeding after food restriction

The SREBP-1c mRNA level and precursor (microsomal) form of SREBP-1 abundance were significantly higher in epididymal and perirenal than in subcutaneous white adipose tissue of control rats. Moreover, the SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were significantly higher in the epididymal and perirenal white adipose tissue of rats maintained on restricted diet and refed ad libitum for 48 h as compared to the control animals. No significant effects of food restriction/refeeding on SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were found in subcutaneous white adipose tissue. The mature (nuclear) form of SREBP-1 was significantly increased in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed animals. The activity, protein level and the mRNA abundance of malic enzyme (one of the target genes for SREBP-1) increased significantly in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed rats as compared to the control animals, however the increase in perirenal and epididymal was higher than in the subcutaneous white adipose tissue. The results presented suggest that SREBP-1c is differently expressed in various rat white adipose tissue depots both under basal (control) and dieting conditions.