Nicotine infusion alters leptin and uncoupling protein 1 mRNA expression in adipose tissues of rats

We attempted to clarify whether leptin and uncoupling protein 1 (UCP1) are involved in the action of nicotine on the energy balance. Male Wistar rats were infused subcutaneously with nicotine (12 mg x kg(-1) x day(-1)) for 4 or 14 days. At the end of the 4-day period, the plasma concentrations of leptin of the nicotine-treated and pair-fed rats were lower than those of the freely fed rats, although the levels of leptin mRNA expression in various white adipose tissues did not differ among the three groups. At the end of the 14-day nicotine infusion period, plasma concentrations of leptin were higher, and leptin mRNA expression in the omentum and epididymal and retroperitoneal adipose tissues was stronger in the nicotine-treated rats than in the pair-fed and freely fed rats. UCP1 mRNA expression in the brown adipose tissue of nicotine-treated was stronger than that of the pair-fed rats. These results suggest that continuous nicotine infusion differentially affects the synthesis and secretion of leptin according to the duration of infusion and stimulates UCP1 mRNA expression, probably in a manner independent of leptin.     

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs beta-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5-22 mmol/l)+/-leptin (0-10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of beta-cell function in diabetes.     

Gene expression, tissue distribution and potential physiological role of uncoupling protein in avian species

Whole-body energy homeostasis and food intake control are essential for an economically sound selection for growth in poultry. The cellular and molecular mechanisms that regulate and link food intake, energy expenditure and energy balance are still poorly understood in poultry. Mitochondrial uncoupling protein-1 (UCP-1) is known to uncouple respiration from ATP synthesis by short circuiting the inward proton flow, resulting in heat production. Its role seems quite well established in adaptive thermogenesis and energy metabolism. However, uncertainty still surrounds the physiological function of the recently discovered UCP-1 homologues, UCP-2 and -3. Most of the functional characterization of these UCPs, to date, has been conducted in mammals. Recently, an avian UCP homologue, which was identified in chicken, hummingbird and king penguin, appears to play a key role in adaptative thermogenesis. Here, we review recent reports describing avian UCP (av-UCP) and discuss progress concerning the molecular mechanisms and potential role of the av-UCP in thermogenesis regulation in avian species.     

An essential role for a protein and lipid kinase complex in secretory protein sorting

Yeast genetics has identified more than 40 genes involved in the biogenesis and maintenance of the yeast lysosome-like vacuole. Recent data on two of these genes, VPS15 and VPS34, are beginning to provide some fundamental insights into the mechanisms governing protein sorting within the eukaryotic secretory pathway. VPS15 and VPS34 encode a novel protein kinase and a phosphatidylinositol 3-kinase, respectively, that function together as components of a membrane-associated signal transduction complex. These studies of the VPS15-VPS34 complex indicate that intracellular protein trafficking decisions may be regulated by protein phosphorylation and phosphatidylinositol signalling events.     

Upregulation in the expression of multidrug resistance protein Mrp1 mRNA and protein by increased bilirubin production in rat

Earlier studies suggest that Mrp1 may mediate ATP-dependent cellular extrusion of unconjugated bilirubin (UCB). We studied the serial responses of expression of Mrp1 mRNA and protein in rats with increased bilirubin production due to hemolysis induced by phenylhydrazine (PHZ) treatment. Mrp1 mRNA was analyzed by quantitative PCR and protein by Western blot. Hepatic expression of Mrp1 mRNA and protein peaked at day 3 of PHZ treatment. Splenic expression of Mrp1 mRNA peaked within 24h and returned to baseline at day 5 whereas Mrp1 protein expression peaked at day 3. Pretreatment with heme-oxygenase inhibitor, tin mesoporphyrin, blunted the increase in serum UCB and erased the overexpression of Mrp1 both in liver and spleen. Thus, the upregulation of Mrp1 in hemolysis is mediated by UCB and/or other products of heme oxygenase, further supporting a role of Mrp1 in UCB transport and protection from its cellular toxicity.     

Dermal connective tissue development in mice: an essential role for tenascin-X

Deficiency of the extracellular matrix protein tenascin-X (TNX) causes a recessive form of Ehlers‐Danlos syndrome (EDS) characterized by hyperextensible skin and hypermobile joints. It is not known whether the observed alterations of dermal collagen fibrils and elastic fibers in these patients are caused by disturbed assembly and deposition or by altered stability and turnover. We used biophysical measurements and immunofluorescence to study connective tissue properties in TNX knockout and wild-type mice. We found that TNX knockout mice, even at a young age, have greatly disturbed biomechanical properties of the skin. No joint abnormalities were noted at any age. The spatio-temporal expression of TNX during normal mouse skin development, during embryonic days 13–19 (E13–E19), was distinct from tropoelastin and the dermal fibrillar collagens type I, III, and V. Our data show that TNX is not involved in the earliest phase (E10–E14) of the deposition of collagen fibrils and elastic fibers during fetal development. From E15 to E19, TNX starts partially to colocalize with the dermal collagens and elastin, and in adult mice, TNX is present in the entire dermis. In adult TNX knockout mice, we observed an apparent increase of elastin. We conclude that TNX knockout mice only partially recapitulate the phenotype of TNX-deficient EDS patients, and that TNX could potentially be involved in maturation and/or maintenance of the dermal collagen and elastin network.     

Derlin-2-deficient mice reveal an essential role for protein dislocation in chondrocytes

Protein quality control is a balance between chaperone-assisted folding and removal of misfolded proteins from the endoplasmic reticulum (ER). Cell-based assays have been used to identify key players of the dislocation machinery, including members of the Derlin family. We generated conditional knockout mice to examine the in vivo role of Derlin-2, a component that nucleates cellular dislocation machinery. In most Derlin-2-deficient tissues, we found constitutive upregulation of ER chaperones and IRE-1-mediated induction of the unfolded protein response. The IRE-1/XBP-1 pathway is required for development of highly secretory cells, particularly plasma cells and hepatocytes. However, B lymphocyte development and antibody secretion were normal in the absence of Derlin-2. Likewise, hepatocyte function was unaffected by liver-specific deletion of Derlin-2. Whole-body deletion of Derlin-2 results in perinatal death. The few mice that survived to adulthood all developed skeletal dysplasia, likely caused by defects in collagen matrix protein secretion by costal chondrocytes.     

Increased uncoupling protein2 mRNA in white adipose tissue, and decrease in leptin, visceral fat, blood glucose, and cholesterol in KK-Ay mice fed with eicosapentaenoic and docosahexaenoic acids in addition to linolenic acid.

The effects of n-3 polyunsaturated fatty acids (n-3PUFA) on obesity and diabetes were examined using KK-Ay mice fed with perilla oil (P), soybean oil (S), or lard (L), and those containing 30% fish oil (PF, SF, or LF), containing eicosapentaenoic acid (EPA = 9.9%) and docosahexaenoic acid (DHA = 18.0%). Perilla oil contained the largest proportion of linolenic acid (LNA = 61.9%). Computerized tomography (CT) scans showed narrower areas of visceral fat in the abdominal cross sections of groups given fish oil (PF, SF, and LF) and lower leptin levels (p < 0.05-p < 0.001) compared with controls (P, S, and L), without significant changes in energy intake and body weight. The highest plasma n-3PUFA content (21.31 +/- 0.35%) was attained with PF. This group contained 2.6-fold more plasma DHA (p < 0.001), and expressed 2.7-fold more UCP2 mRNA in white adipose tissue (p < 0.01) than in the P group. The epididymal fat pad (p < 0.05) weighed less, and levels of blood glucose (p < 0.05) and total cholesterol (p < 0.01) were reduced in PF compared with P.     

Seasonal regulations of energetics, serum concentrations of leptin, and uncoupling protein 1 content of brown adipose tissue in root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau

Survival of small mammals in winter requires proper adjustments in physiology, behavior and morphology. The present study was designed to examine the changes in serum leptin concentration and the molecular basis of thermogenesis in seasonally acclimatized root voles (Microtus oeconomus) from the Qinghai-Tibetan plateau. In January root voles had lower body mass and body fat mass coupled with higher nonshivering thermogenesis (NST) capacity. Consistently, cytochrome c oxidase activity and mitochondrial uncoupling protein-1 (UCP1) protein contents in brown adipose tissues were higher in January as compared to that in July. Circulating level of serum leptin was significantly lower in winter and higher in July. Correlation analysis showed that serum leptin levels were positively related with body mass and body fat mass while negatively correlated with UCP1 protein contents. Together, these data provided further evidence for our previous findings that root voles from the Qinghai-Tibetan plateau mainly depend on higher NST coupled with lower body mass to enhance winter survival. Further, fat deposition was significantly mobilized in cold winter and leptin was potentially involved in the regulation of body mass and thermogenesis in root voles. Serum leptin might act as a starvation signal in winter and satiety signal in summer.     

A role for NPY overexpression in the dorsomedial hypothalamus in hyperphagia and obesity of OLETF rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors are hyperphagic, obese, and diabetic. We have previously demonstrated that these rats have a peripheral satiety deficit resulting in increased meal size. To examine the potential role of hypothalamic pathways in the hyperphagia and obesity of OLETF rats, we compared patterns of hypothalamic neuropeptide Y (NPY), proopiomelanocortin (POMC), and leptin receptor mRNA expression in ad libitum-fed Long-Evans Tokushima (LETO) and OLETF rats and food-restricted OLETF rats that were pair-fed to the intake of LETO controls. Pair feeding OLETF rats prevented their increased body weight and elevated levels of plasma insulin and leptin and normalized their elevated POMC and decreased NPY mRNA expression in the arcuate nucleus. In contrast, NPY expression was upregulated in the dorsomedial hypothalamus (DMH) in pair-fed OLETF rats. A similar DMH NPY overexpression was evident in 5-wk-old preobese OLETF rats. These findings suggest a role for DMH NPY upregulation in the etiology of OLETF hyperphagia and obesity.     

The role of uncoupling protein 2 in macrophages and its impact on obesity-induced adipose tissue inflammation and insulin resistance

The development of a chronic, low-grade inflammation originating from adipose tissue in obese subjects is widely recognized to induce insulin resistance, leading to the development of type 2 diabetes. The adipose tissue microenvironment drives specific metabolic reprogramming of adipose tissue macrophages, contributing to the induction of tissue inflammation. Uncoupling protein 2 (UCP2), a mitochondrial anion carrier, is thought to separately modulate inflammatory and metabolic processes in macrophages and is up-regulated in macrophages in the context of obesity and diabetes. Here, we investigate the role of UCP2 in macrophage activation in the context of obesity-induced adipose tissue inflammation and insulin resistance. Using a myeloid-specific knockout of UCP2 (Ucp2^ΔLysM), we found that UCP2 deficiency significantly increases glycolysis and oxidative respiration, both unstimulated and after inflammatory conditions. Strikingly, fatty acid loading abolished the metabolic differences between Ucp2^ΔLysM macrophages and their floxed controls. Furthermore, Ucp2^ΔLysM macrophages show attenuated pro-inflammatory responses toward Toll-like receptor-2 and -4 stimulation. To test the relevance of macrophage-specific Ucp2 deletion in vivo, Ucp2^ΔLysM and Ucp2^fl/fl mice were rendered obese and insulin resistant through high-fat feeding. Although no differences in adipose tissue inflammation or insulin resistance was found between the two genotypes, adipose tissue macrophages isolated from diet-induced obese Ucp2^ΔLysM mice showed decreased TNFα secretion after ex vivo lipopolysaccharide stimulation compared with their Ucp2^fl/fl littermates. Together, these results demonstrate that although UCP2 regulates both metabolism and the inflammatory response of macrophages, its activity is not crucial in shaping macrophage activation in the adipose tissue during obesity-induced insulin resistance.     

Hypolipidemic effects of crude extract of adlay seed (Coix lachrymajobi var. mayuen) in obesity rat fed high fat diet: relations of TNF-alpha and leptin mRNA expressions and serum lipid levels

To find out whether the expressions of these adipocyte markers are influenced by oriental medicine, obesity rats induced by high fat diet (HFD) for 8 weeks were injected with 50 mg/100 g body weight adlay seed crude extract (ACE), daily for 4 weeks. The results are summarized as follows: HFD + ACE group significantly reduced food intakes and body weights. Weights of epididymal and peritoneal fat were dramatically increased in HFD groups compared with those of normal diet (ND) group but significantly decreased more in HFD + ACE group than those of HFD + saline group (sham). Those of brown adipocytes were increased in HFD + ACE group compared to ND and sham groups but there was no significant difference. The sizes in white adipose tissue (WAT) by microscope were markedly larger in HFD groups than ND group but considerably reduced in HFD + ACE group compared with sham group. The levels of triglyceride, total-cholesterol and leptin in blood serum were significantly decreased in HFD + ACE group compared to those of sham group. Leptin and TNF-alpha mRNA expressions in WAT of rats were remarkably increased more in sham group than in those of ND group. Those of HFD + ACE group were significantly decreased compared with those of sham group, especially. TNF-alpha mRNA expression in HFD + ACE group was declined more than that of ND group. In conclusion, treatments of ACE modulated expressions of leptin and TNF-alpha and reduced body weights, food intake, fat size, adipose tissue mass and serum hyperlipidemia in obesity rat fed HFD. Accordingly, the oriental medicine extract, adlay seed crude extract, can be considered for obesity therapies controlling.     

Visfatin, TNF-alpha and IL-6 mRNA expression is increased in mononuclear cells from type 2 diabetic women.

Visfatin, is a new adipokine, highly expressed in the visceral fat of both mice and humans. To examine whether visfatin is expressed in human peripheral monocyte-enriched mononuclear cells and whether its expression is altered in type 2 diabetes (DM2), we compared 24 DM2 women [17 overweight (BMI >25) and 7 lean (BMI<25)] to 26 healthy women (14 overweight and 12 lean), all premenopausal. Relative visfatin mRNA levels were significantly higher (approximately 3-fold) in DM2 compared to healthy control women (p<0.02), independently of the presence of overweight/obesity. Mononuclear TNF-alpha and IL-6 mRNA expression was also elevated in DM2 compared to control women (p=0.001 and p=0.004, respectively), an increase observed in both lean and overweight DM2 women. By contrast, circulating visfatin, TNF-alpha, and IL-6 levels showed no difference between DM2 and control women, while adiponectin plasma levels were significantly decreased in the DM2 women (p<0.001). Circulating visfatin and TNF-alpha levels did not differ either between the lean and the overweight subgroups of DM2 and control women, while IL-6 plasma levels were significantly higher in both overweight subgroups compared to their lean counterparts. In conclusion, visfatin, TNF-alpha, and IL-6 mRNA expressions are increased in peripheral mononuclear-monocytic cells from women with type 2 diabetes, independent of their BMI, which may enhance the effects of their adipose-derived levels and may contribute to the increased insulin resistance and atherogenic risk of these patients.     

Increase of uncoupling protein and its mRNA in brown adipose tissue of rats fed on ''cafeteria diet''.

The effect of 'cafeteria diet' on mitochondrial uncoupling protein in brown adipose tissue of rats was examined. 'Cafeteria diet' induced an increase of the 32 kDa uncoupling protein in electrophoresed proteins of brown-fat mitochondria. Use of a cDNA probe corresponding to uncoupling-protein mRNA indicated that this mRNA was increased in rats fed on the 'cafeteria diet'. Nevertheless, this effect was weak compared with that observed in rats adapted to cold.     

Attenuated cold-induced increase in mRNA for uncoupling protein in brown adipose tissue of obese (ob/ob) mice

The level of mRNA for uncoupling protein was measured in brown adipose tissue of young (8-10 weeks) and old (11 months) lean and ob/ob mice using a cDNA clone constructed previously. The level of poly(A)+ RNA was also measured using an oligo(dT)18 probe. Mice were kept at 28 degrees C or exposed to 14 degrees C for 12 h. The level of mRNA for uncoupling protein was normal in brown adipose tissue of younger obese mice but reduced in brown adipose tissue of old obese mice. The cold-induced absolute increase in uncoupling protein mRNA was smaller in obese mice, regardless of age. It is concluded that the known attenuation of the acute thermogenic response of brown adipose tissue of the ob/ob mouse to cold is accompanied by a similar attenuation of the initiation of the trophic response. It is likely, however, that these defects are secondary to the chronic reduction in sympathetic nervous system activity in brown adipose tissue of the ob/ob mouse, which results in a functional atrophy of the tissue.     

The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of Os Tudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. Os Tudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that Os Tudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that Os Tudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of Os Tudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of Os Tudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged Os Tudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of Os Tudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that Os Tudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.     

Activation of AMP-activated protein kinase is required for berberine-induced reduction of atherosclerosis in mice: the role of uncoupling protein 2

Aims

Berberine, a botanical alkaloid purified from Coptidis rhizoma, is reported to activate the AMP-activated protein kinase (AMPK). Whether AMPK is required for the protective effects of berberine in cardiovascular diseases remains unknown. This study was designed to determine whether AMPK is required for berberine-induced reduction of oxidative stress and atherosclerosis in vivo.

Methods

ApoE (ApoE^-/-) mice and ApoE^-/-/AMPK alpha 2^-/- mice that were fed Western diets were treated with berberine for 8 weeks. Atherosclerotic aortic lesions, expression of uncoupling protein 2 (UCP2), and markers of oxidative stress were evaluated in isolated aortas.

Results

In ApoE^-/- mice, chronic administration of berberine significantly reduced aortic lesions, markedly reduced oxidative stress and expression of adhesion molecules in aorta, and significantly increased UCP2 levels. In contrast, in ApoE^-/-/AMPK alpha 2^-/- mice, berberine had little effect on those endpoints. In cultured human umbilical vein endothelial cells (HUVECs), berberine significantly increased UCP2 mRNA and protein expression in an AMPK-dependent manner. Transfection of HUVECs with nuclear respiratory factor 1 (NRF1)-specific siRNA attenuated berberine-induced expression of UCP2, whereas transfection with control siRNA did not. Finally, berberine promoted mitochondrial biogenesis that contributed to up-regulation of UCP2 expression.

Conclusion

We conclude that berberine reduces oxidative stress and vascular inflammation, and suppresses atherogenesis via a mechanism that includes stimulation of AMPK-dependent UCP2 expression.