Ablation of PGC1 beta prevents mTOR dependent endoplasmic reticulum stress response.

Mitochondria dysfunction contributes to the pathophysiology of obesity, diabetes, neurodegeneration and ageing. The peroxisome proliferator-activated receptor-gamma coactivator-1ß (PGC-1ß) coordinates mitochondrial biogenesis and function as well as fatty acid metabolism. It has been suggested that endoplasmic reticulum (ER) stress may be one of the mechanisms linking mitochondrial dysfunction and these pathologies. Here we investigate whether PGC-1ß ablation affects the ER stress response induced by specific nutritional and pharmacological challenges in the CNS. By using flow cytometry, western blot, real time PCR and several pharmacological and nutritional interventions in PGC-1ß knock out and WT mice, we confirmed that PGC-1ß coordinates mitochondria function in brain and reported for the first time that a) ablation of PGC-1ß is associated with constitutive activation of mTORC1 pathway associated with increased basal GRP78 protein levels in hypothalamus and cortex of animals fed chow diet; and b) in animals fed chronically with high fat diet (HFD) or high protein diet (HPD), we observed a failure to appropriately induce ER stress response in the absence of PGC-1ß, associated with an increase in mTOR pathway phosphorylation. This contrasted with the appropriate upregulation of ER stress response observed in wild type littermates. Additionally, inefficient in vitro induction of ER stress by thapsigargin seems result in apoptotic neuronal cell death in PGC-1ß KO. Our data indicate that PGC-1ß is required for a neuronal ER response to nutritional stress imposed by HFD and HPD diets and that genetic ablation of PGC-1ß might increase the susceptibility to neuronal damage and cell death.

Lipocalin prostaglandin D synthase and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism in vivo.

Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism.

Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans.

Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence-binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a complex phenotype. Azoospermia, with failure of the latter stages of spermatogenesis, was associated with a lack of testis-enriched selenoproteins. An axial muscular dystrophy was also present, with features similar to myopathies caused by mutations in selenoprotein N (SEPN1). Cutaneous deficiencies of antioxidant selenoenzymes, increased cellular ROS, and susceptibility to ultraviolet radiation-induced oxidative damage may mediate the observed photosensitivity. Reduced levels of selenoproteins in peripheral blood cells were associated with impaired T lymphocyte proliferation, abnormal mononuclear cell cytokine secretion, and telomere shortening. Paradoxically, raised ROS in affected subjects was associated with enhanced systemic and cellular insulin sensitivity, similar to findings in mice lacking the antioxidant selenoenzyme glutathione peroxidase 1 (GPx1). Thus, mutation of SECISBP2 is associated with a multisystem disorder with defective biosynthesis of many selenoproteins, highlighting their role in diverse biological processes.

Adaptation and failure of pancreatic beta cells in murine models with different degrees of metabolic syndrome.

The events that contribute to the expansion of beta-cell mass and enhanced beta-cell function in insulin-resistant states have not been elucidated fully. Recently, we showed that beta-cell adaptation failed dramatically in adult, insulin-resistant POKO mice, which contrasts with the appropriate expansion of beta cells in their ob/ob littermates. Thus, we hypothesised that characterisation of the islets in these mouse models at an early age should provide a unique opportunity to: (1) identify mechanisms involved in sensing insulin resistance at the level of the beta cells, (2) identify molecular effectors that contribute to increasing beta-cell mass and function, and (3) distinguish primary events from secondary events that are more likely to be present at more advanced stages of diabetes. Our results define the POKO mouse as a model of early lipotoxicity. At 4 weeks of age, it manifests with inappropriate beta-cell function and defects in proliferation markers. Other well-recognised pathogenic effectors that were observed previously in 16-week-old mice, such as increased reactive oxygen species (ROS), macrophage infiltration and endoplasmic reticulum (ER) stress, are also present in both young POKO and young ob/ob mice, indicating the lack of predictive power with regards to the severity of beta-cell failure. Of interest, the relatively preserved lipidomic profile in islets from young POKO mice contrasted with the large changes in lipid composition and the differences in the chain length of triacylglycerols in the serum, liver, muscle and adipose tissue in adult POKO mice. Later lipotoxic insults in adult beta cells contribute to the failure of the POKO beta cell. Our results indicate that the rapid development of insulin resistance and beta-cell failure in POKO mice makes this model a useful tool to study early molecular events leading to insulin resistance and beta-cell failure. Furthermore, comparisons with ob/ob mice might reveal important adaptive mechanisms in beta cells with either therapeutic or diagnostic potential.

Defective peroxisomal proliferators activated receptor gamma activity due to dominant-negative mutation synergizes with hypertension to accelerate cardiac fibrosis in mice.

AIMS: Humans with inactivating mutations in peroxisomal proliferators activated receptor gamma (PPARgamma) typically develop a complex metabolic syndrome characterized by insulin resistance, diabetes, lipodystrophy, hypertension, and dyslipidaemia which is likely to increase their cardiovascular risk. Despite evidence that the activation of PPARgamma may prevent cardiac fibrosis and hypertrophy, recent evidence has suggested that pharmacological activation of PPARgamma causes increased cardiovascular mortality. In this study, we investigated the effects of defective PPARgamma function on the development of cardiac fibrosis and hypertrophy in a murine model carrying a human dominant-negative mutation in PPARgamma. METHODS AND RESULTS: Mice with a dominant-negative point mutation in PPARgamma (P465L) and their wild-type (WT) littermates were treated with either subcutaneous angiotensin II (AngII) infusion or saline for 2 weeks. Heterozygous P465L and WT mice developed a similar increase in systolic blood pressure, but the mutant mice developed significantly more severe cardiac fibrosis to AngII that correlated with increased expression of profibrotic genes. Both groups similarly increased the heart weight to body weight ratio compared with saline-treated controls. There were no differences in fibrosis between saline-treated WT and P465L mice. CONCLUSION: These results show synergistic pathogenic effects between the presence of defective PPARgamma and AngII-induced hypertension and suggest that patients with PPARgamma mutation and hypertension may need more aggressive therapeutic measures to reduce the risk of accelerated cardiac fibrosis.

PPAR gamma 2 prevents lipotoxicity by controlling adipose tissue expandability and peripheral lipid metabolism.

Peroxisome proliferator activated receptor gamma 2 (PPARg2) is the nutritionally regulated isoform of PPARg. Ablation of PPARg2 in the ob/ob background, PPARg2(-/-) Lep(ob)/Lep(ob) (POKO mouse), resulted in decreased fat mass, severe insulin resistance, beta-cell failure, and dyslipidaemia. Our results indicate that the PPARg2 isoform plays an important role, mediating adipose tissue expansion in response to positive energy balance. Lipidomic analyses suggest that PPARg2 plays an important antilipotoxic role when induced ectopically in liver and muscle by facilitating deposition of fat as relatively harmless triacylglycerol species and thus preventing accumulation of reactive lipid species. Our data also indicate that PPARg2 may be required for the beta-cell hypertrophic adaptive response to insulin resistance. In summary, the PPARg2 isoform prevents lipotoxicity by (a) promoting adipose tissue expansion, (b) increasing the lipid-buffering capacity of peripheral organs, and (c) facilitating the adaptive proliferative response of beta-cells to insulin resistance.

Ablation of PGC-1beta results in defective mitochondrial activity, thermogenesis, hepatic function, and cardiac performance.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta) has been implicated in important metabolic processes. A mouse lacking PGC-1beta (PGC1betaKO) was generated and phenotyped using physiological, molecular, and bioinformatic approaches. PGC1betaKO mice are generally viable and metabolically healthy. Using systems biology, we identified a general defect in the expression of genes involved in mitochondrial function and, specifically, the electron transport chain. This defect correlated with reduced mitochondrial volume fraction in soleus muscle and heart, but not brown adipose tissue (BAT). Under ambient temperature conditions, PGC-1beta ablation was partially compensated by up-regulation of PGC-1alpha in BAT and white adipose tissue (WAT) that lead to increased thermogenesis, reduced body weight, and reduced fat mass. Despite their decreased fat mass, PGC1betaKO mice had hypertrophic adipocytes in WAT. The thermogenic role of PGC-1beta was identified in thermoneutral and cold-adapted conditions by inadequate responses to norepinephrine injection. Furthermore, PGC1betaKO hearts showed a blunted chronotropic response to dobutamine stimulation, and isolated soleus muscle fibres from PGC1betaKO mice have impaired mitochondrial function. Lack of PGC-1beta also impaired hepatic lipid metabolism in response to acute high fat dietary loads, resulting in hepatic steatosis and reduced lipoprotein-associated triglyceride and cholesterol content. Altogether, our data suggest that PGC-1beta plays a general role in controlling basal mitochondrial function and also participates in tissue-specific adaptive responses during metabolic stress.

Tamoxifen-induced anorexia is associated with fatty acid synthase inhibition in the ventromedial nucleus of the hypothalamus and accumulation of malonyl-CoA.

Fatty acid metabolism in the hypothalamus has recently been shown to regulate feeding. The selective estrogen receptor modulator tamoxifen (TMX) exerts a potent anorectic effect. Here, we show that the anorectic effect of TMX is associated with the accumulation of malonyl-CoA in the hypothalamus and inhibition of fatty acid synthase (FAS) expression specifically in the ventromedial nucleus of the hypothalamus (VMN). Furthermore, we demonstrate that FAS mRNA expression is physiologically regulated by fasting and refeeding in the VMN but not in other hypothalamic nuclei. Thus, the VMN appears to be the hypothalamic site where regulation of FAS and feeding converge. Supporting the potential clinical relevance of these observations, reanalysis of a primary breast cancer prevention study showed that obese women treated with TMX gained significantly less body weight over a 6-year period than obese women given placebo. The finding that TMX can modulate appetite through alterations in FAS expression and malonyl-CoA levels suggests a link between hypothalamic sex steroid receptors, fatty acid metabolism, and feeding behavior.

The link between nutritional status and insulin sensitivity is dependent on the adipocyte-specific peroxisome proliferator-activated receptor-gamma2 isoform.

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is critically required for adipogenesis. PPARgamma exists as two isoforms, gamma1 and gamma2. PPARgamma2 is the more potent adipogenic isoform in vitro and is normally restricted to adipose tissues, where it is regulated more by nutritional state than PPARgamma1. To elucidate the relevance of the PPARgamma2 in vivo, we generated a mouse model in which the PPARgamma2 isoform was specifically disrupted. Despite similar weight, body composition, food intake, energy expenditure, and adipose tissue morphology, male mice lacking the gamma2 isoform were more insulin resistant than wild-type animals when fed a regular diet. These results indicate that insulin resistance associated with ablation of PPARgamma2 is not the result of lipodystrophy and suggests a specific role for PPARgamma2 in maintaining insulin sensitivity independently of its effects on adipogenesis. Furthermore, PPARgamma2 knockout mice fed a high-fat diet did not become more insulin resistant than those on a normal diet, despite a marked increase in their mean adipocyte cell size. These findings suggest that PPARgamma2 is required for the maintenance of normal insulin sensitivity in mice but also raises the intriguing notion that PPARgamma2 may be necessary for the adverse effects of a high-fat diet on carbohydrate metabolism.

Dopa oxidase activity in the hair, skin and ocular melanocytes is increased in the presence of stressed fibroblasts.

We previously reported that mesenchymal cells (dermal fibroblasts and dermal papilla cells) can stimulate dopa oxidase activity in the skin melanocytes. This study extends the investigation of the influence of the fibroblast in a comparative study of melanogenesis in melanocytes from the hair, the skin and the eye. Culture of melanocytes with normal proliferative dermal fibroblasts slightly increased dopa oxidase activity of the hair, skin and ocular melanocytes (by 17, 11 and 28%, respectively), but co-culture with fibroblasts recovering from storage in liquid nitrogen or growth-arrested by means of gamma radiation showed much greater effects. Most dramatic results were obtained with fibroblasts, which had been both gamma-irradiated and then frozen in liquid nitrogen, where increases in dopa oxidase activity of 125, 227 and 185% for melanocytes of the hair, the skin and the eye, respectively, were seen. Experiments by using transwell cultures of melanocytes and fibroblasts and by using fibroblast-conditioned medium showed that a large proportion of this fibroblast influence could be mediated by diffusible factors, of which a good proportion was attributable to basic Fibroblast Growth Factor (bFGF). The addition of bFGF significantly increased dopa oxidase activity of the skin melanocytes, when fibroblasts were present, but not in their absence. These data show that fibroblasts in vitro, particularly when deliberately stressed, have the ability to increase dopa oxidase activity in melanocytes of the hair, the skin and the eye and further suggest that this effect is mediated by bFGF acting in combination with some other fibroblast-derived factors.

Ethnic variation in tyrosinase and TYRP1 expression in photoexposed and photoprotected human skin.

The relative expression of a number of key mediators of human pigmentation including tyrosinase, tyrosinase related protein-1 (TYRP1), endothelin-1 and adrenocorticotrophic hormone (ACTH) proteins were analysed and quantified in immunohistochemically stained skin sections using semiquantitative computer assisted image analysis. Comparisons were made between a range of different ethnic skin types including European, Chinese, Mexican, Indian and African at both chronically photoexposed and photoprotected sites. Melanocyte number varied little with ethnicity except in the European group which had 60-80% more melanocytes than other skin types (P < 0.01, n = 10; Student Neuman-Keuls). However, melanocyte number was increased approximately twofold in chronically photoexposed skin of all ethnic groups (P < 0.001, n = 48; paired t-test). Tyrosinase protein expression in melanocytes did not vary with ethnicity, but TYRP1 protein was significantly elevated (approximately 2.6-fold) in darkly pigmented African and Indian skin types compared with lightly pigmented Mexican, Chinese and European skin types. In melanocytes from chronically photoexposed skin, there was a modest but significant increase in the expression of tyrosinase protein (approximately 1.2-fold, P < 0.001, n = 48; paired t-test), together with a significant and slightly larger increase in the expression of TYRP1 protein (approximately 1.6-fold, P < 0.005, n = 48; paired t-test). In contrast, the expression of endothelin-1 and ACTH showed no significant variation with either ethnicity or photoexposure. These data are consistent with the view that maintenance of a chronically hyperpigmented phenotype in chronically photoexposed human skin is largely the result of a stable increase in the number of tyrosinase positive melanocytes at these sites. Moreover, the observed ethnic variation in TYRP1 protein expression suggests that TYRP1 may play a significant role in mediating ethnic differences in melanogenesis and constitutive skin pigmentation in vivo.

Ethnic variation in melanin content and composition in photoexposed and photoprotected human skin.

We have examined the quantity and composition of melanin in both photoprotected (volar upper arm) and chronically photoexposed (dorsal forearm) skin from a range of different ethnic skin types including African, Indian, Mexican, Chinese and European. The most lightly pigmented (European, Chinese and Mexican) skin types have approximately half as much epidermal melanin as the most darkly pigmented (African and Indian) skin types. However, the composition of melanin in these lighter skin types is comparatively more enriched with lightly coloured, alkali-soluble melanin components (up to three-fold). Regardless of ethnicity, epidermal melanin content is significantly greater in chronically photoexposed skin than it is in corresponding photoprotected skin (up to two-fold). However, by comparison there is only a modest enrichment of lightly coloured, alkali soluble melanin components in photoprotected skin (up to 1.3-fold). Analysis of melanosomes extracted from the epidermis in these subjects indicates that the proportion of spheroidal melanosomes is low in all skin types examined (<10%). This suggests that in human skin, pheomelanin is a very minor component of epidermal melanin, even in the lightest (European) skin types. Analysis of melanosome size revealed a significant and progressive variation in size with ethnicity: African skin having the largest melanosomes followed in turn by Indian, Mexican, Chinese and European. On the basis of these findings, we propose that variation in skin pigmentation is strongly influenced by both the amount and the composition (or colour) of the melanin in the epidermis. Variation in melanosome size may also play a significant role. However, the data also suggest that in human skin there are subtle differences in the mechanisms associated with the maintenance of constitutive pigmentation and facultative hyperpigmentation, respectively.

The impact of epidermal melanin on objective measurements of human skin colour.

Objective measurements of human skin colour were made with a tristimulus (L*a*b*) chromameter in a range of different ethnic skin types. These were compared with biochemical measurements of melanin content, melanin composition and melanosome size in skin biopsies obtained from the same sites. L*, a* and b* values were found to vary significantly with ethnicity. In general, constitutively dark skin types have lower L* values, higher a* values and higher b* values than constitutively light skin types. Total epidermal melanin content appears to be the primary determinant of L* values in human skin (r = -0.88; P < 0.00001), whilst melanosome size also has a significant but more subtle influence on L* values (r = -0.73; P < 0.00001). There is also a strong positive contribution to a* values from epidermal melanin (r = 0.66, P < 0.00001), which accounts for the ethnic variation in a* values observed in this study. Melanin is also a major contributor to b* values in lighter skin types (r = 0.71, P < 0.00001). However, this relationship breaks down in darker skin types where b* values actually reach a maximum and then decrease as the concentration of melanin in the skin increases. This appears to be because of optical masking of yellow light by high concentrations of melanin in the epidermis. Analysis of the relationships between L*, a* and b* values in human skin indicate that they are very closely interrelated, and suggest that the optical properties of melanin in the epidermis are very similar to those of a dye on a fabric substrate.