Rhes, a striatal-enriched protein, promotes mitophagy via Nix.

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here, we find that striatal-enriched protein, Rhes, is a critical regulator of mitophagy and striatal vulnerability in brain. In vivo interactome and density fractionation reveal that Rhes coimmunoprecipitates and cosediments with mitochondrial and lysosomal proteins. Live-cell imaging of cultured striatal neuronal cell line shows Rhes surrounds globular mitochondria, recruits lysosomes, and ultimately degrades mitochondria. In the presence of 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, Rhes disrupts mitochondrial membrane potential (ΔΨ m ) and promotes excessive mitophagy and cell death. Ultrastructural analysis reveals that systemic injection of 3-NP in mice promotes globular mitochondria, accumulation of mitophagosomes, and striatal lesion only in the wild-type (WT), but not in the Rhes knockout (KO), striatum, suggesting that Rhes is critical for mitophagy and neuronal death in vivo. Mechanistically, Rhes requires Nix (BNIP3L), a known receptor of mitophagy, to disrupt ΔΨ m and promote mitophagy and cell death. Rhes interacts with Nix via SUMO E3-ligase domain, and Nix depletion totally abrogates Rhes-mediated mitophagy and cell death in the cultured striatal neuronal cell line. Finally, we find that Rhes, which travels from cell to cell via tunneling nanotube (TNT)-like cellular protrusions, interacts with dysfunctional mitochondria in the neighboring cell in a Nix-dependent manner. Collectively, Rhes is a major regulator of mitophagy via Nix, which may determine striatal vulnerability in the brain.

Comprehensive molecular characterization of human adipocytes reveals a transient brown phenotype.

BACKGROUND: Functional brown adipose tissue (BAT), involved in energy expenditure, has recently been detected in substantial amounts in adults. Formerly overlooked BAT has now become an attractive anti-obesity target. METHODS AND RESULTS: Molecular characterization of human brown and white adipocytes, using a myriad of techniques including high-throughput RNA sequencing and functional assays, showed that PAZ6 and SW872 cells exhibit classical molecular and phenotypic markers of brown and white adipocytes, respectively. However, the pre-adipocyte cell line SGBS presents a versatile phenotype. A transit expression of classical brown markers such as UCP1 and PPARγ peaked and declined at day 28 post-differentiation initiation. Conversely, white adipocyte markers, including Tcf21, showed reciprocal behavior. Interestingly, leptin levels peaked at day 28 whereas the highest adiponectin mRNA levels were detected at day 14 of differentiation. Phenotypic analysis of the abundance and shape of lipid droplets were consistent with the molecular patterns. Accordingly, the oxidative capacity of SGBS adipocytes peaked on differentiation day 14 and declined progressively towards differentiation day 28. CONCLUSIONS: Our studies have unveiled a new phenotype of human adipocytes, providing a tool to identify molecular gene expression patterns and pathways involved in the conversion between white and brown adipocytes.

Fatty acid transport proteins, implications in physiology and disease.

Uptake of long-chain fatty acids plays pivotal roles in metabolic homeostasis and human physiology. Uptake rates must be controlled in an organ-specific fashion to balance storage with metabolic needs during transitions between fasted and fed states. Many obesity-associated diseases, such as insulin resistance in skeletal muscle, cardiac lipotoxicity, and hepatic steatosis, are thought to be driven by the overflow of fatty acids from adipose stores and the subsequent ectopic accumulation of lipids resulting in apoptosis, ER stress, and inactivation of the insulin receptor signaling cascade. Thus, it is of critical importance to understand the components that regulate the flux of fatty acid between the different organ systems. Cellular uptake of fatty acids by key metabolic organs, including the intestine, adipose tissue, muscle, heart, and liver, has been shown to be protein mediated and various unique combinations of fatty acid transport proteins (FATPs/SLC27A1-6) are expressed by all of these tissues. Here we review our current understanding of how FATPs can contribute to normal physiology and how FATP mutations as well as hypo- and hypermorphic changes contribute to disorders ranging from cardiac lipotoxicity to hepatosteatosis and ichthyosis. Ultimately, our increasing knowledge of FATP biology has the potential to lead to the development of new diagnostic tools and treatment options for some of the most pervasive chronic human disorders. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

Specific bile acids inhibit hepatic fatty acid uptake in mice.

UNLABELLED: Bile acids are known to play important roles as detergents in the absorption of hydrophobic nutrients and as signaling molecules in the regulation of metabolism. We tested the novel hypothesis that naturally occurring bile acids interfere with protein-mediated hepatic long chain free fatty acid (LCFA) uptake. To this end, stable cell lines expressing fatty acid transporters as well as primary hepatocytes from mouse and human livers were incubated with primary and secondary bile acids to determine their effects on LCFA uptake rates. We identified ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) as the two most potent inhibitors of the liver-specific fatty acid transport protein 5 (FATP5). Both UDCA and DCA were able to inhibit LCFA uptake by primary hepatocytes in a FATP5-dependent manner. Subsequently, mice were treated with these secondary bile acids in vivo to assess their ability to inhibit diet-induced hepatic triglyceride accumulation. Administration of DCA in vivo via injection or as part of a high-fat diet significantly inhibited hepatic fatty acid uptake and reduced liver triglycerides by more than 50%. CONCLUSION: The data demonstrate a novel role for specific bile acids, and the secondary bile acid DCA in particular, in the regulation of hepatic LCFA uptake. The results illuminate a previously unappreciated means by which specific bile acids, such as UDCA and DCA, can impact hepatic triglyceride metabolism and may lead to novel approaches to combat obesity-associated fatty liver disease.

Synthetic ERRα/ß/γ Agonist Induces an ERRα-Dependent Acute Aerobic Exercise Response and Enhances Exercise Capacity.

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and is effective for the prevention and treatment of several diseases. Genetic evidence indicates that the orphan nuclear receptors estrogen receptor-related receptors (ERRs) play an important role in skeletal muscle exercise capacity. Three ERR subtypes exist (ERRα, ß, and γ), and although ERRß/γ agonists have been designed, there have been significant difficulties in designing compounds with ERRα agonist activity. Additionally, there are limited synthetic agonists that can be used to target ERRs in vivo. Here, we report the identification of a synthetic ERR pan agonist, SLU-PP-332, that targets all three ERRs but has the highest potency for ERRα. Additionally, SLU-PP-332 has sufficient pharmacokinetic properties to be used as an in vivo chemical tool. SLU-PP-332 increases mitochondrial function and cellular respiration in a skeletal muscle cell line. When administered to mice, SLU-PP-332 increased the type IIa oxidative skeletal muscle fibers and enhanced exercise endurance. We also observed that SLU-PP-332 induced an ERRα-specific acute aerobic exercise genetic program, and the ERRα activation was critical for enhancing exercise endurance in mice. These data indicate the feasibility of targeting ERRα for the development of compounds that act as exercise mimetics that may be effective in the treatment of numerous metabolic disorders and to improve muscle function in the aging.

Increased insulin sensitivity and diminished pancreatic beta-cell function in DNA repair deficient Ercc1d/- mice.

BACKGROUND: Type 2 diabetes (T2DM) is an age-associated disease characterized by hyperglycemia due to insulin resistance and decreased beta-cell function. DNA damage accumulation has been associated with T2DM, but whether DNA damage plays a role in the pathogenesis of the disease is unclear. Here, we used mice deficient for the DNA excision-repair gene Ercc1 to study the impact of persistent endogenous DNA damage accumulation on energy metabolism, glucose homeostasis and beta-cell function. METHODS: ERCC1-XPF is an endonuclease required for multiple DNA repair pathways and reduced expression of ERCC1-XPF causes accelerated accumulation of unrepaired endogenous DNA damage and accelerated aging in humans and mice. In this study, energy metabolism, glucose metabolism, beta-cell function and insulin sensitivity were studied in Ercc1d/- mice, which model a human progeroid syndrome. RESULTS: Ercc1d/- mice displayed suppression of the somatotropic axis and altered energy metabolism. Insulin sensitivity was increased, whereas, plasma insulin levels were decreased in Ercc1d/- mice. Fasting induced hypoglycemia in Ercc1d/- mice, which was the result of increased glucose disposal. Ercc1d/- mice exhibit a significantly reduced beta-cell area, even compared to control mice of similar weight. Glucose-stimulated insulin secretion in vivo was decreased in Ercc1d/- mice. Islets isolated from Ercc1d/- mice showed increased DNA damage markers, decreased glucose-stimulated insulin secretion and increased susceptibility to apoptosis. CONCLUSION: Spontaneous DNA damage accumulation triggers an adaptive response resulting in improved insulin sensitivity. Loss of DNA repair, however, does negatively impacts beta-cell survival and function in Ercc1d/- mice.

Compositional analysis and biological characterization of Cornus officinalis on human 1.1B4 pancreatic ß cells.

Type 1 diabetes (T1D) is an autoimmune disease resulting from the loss of pancreatic ß cells and subsequent insulin production. Novel interventional therapies are urgently needed that can protect existing ß cells from cytokine-induced death and enhance their function before symptomatic onset. Our initial evidence is suggesting that bioactive ingredients within Cornus officinalis (CO) may be able to serve in this function. CO has been extensively used in Traditional Chinese Medicine (TCM) and reported to possess both anti-inflammatory and pro-metabolic effects. We hypothesize that CO treatment may provide a future potential candidate for interventional therapy for early stage T1D prior to significant ß cell loss. Our data demonstrated that CO can inhibit cytokine-mediated ß cell death, increase cell viability and oxidative capacity, and increase expression of NFATC2 (Nuclear Factor of Activated T Cells, Cytoplasmic 2). We have also profiled the bioactive components in CO from multiple sources by HPLC/MS (High Performance Liquid Chromatography/Mass Spectrometry) analysis. Altogether, CO significantly increases the energy metabolism of ß cells while inducing the NFAT pathway to signal for increased proliferation and endocrine function.

Distinct roles for REV-ERBα and REV-ERBß in oxidative capacity and mitochondrial biogenesis in skeletal muscle.

The nuclear receptors REV-ERBα and REV-ERBß have been demonstrated to be core members of the circadian clock and participate in the regulation of a diverse set of metabolic functions. Due to their overlapping tissue expression patterns and gene expression profiles, REV-ERBß is thought to be redundant to REV-ERBα. Recent work has highlighted REV-ERBα's role in the regulation of skeletal muscle oxidative capacity and mitochondrial biogenesis. Considering the similarity between the REV-ERBs and the hypothesized overlap in function, we sought to determine whether REV-ERBß-deficiency presented with a similar skeletal muscle phenotype as REV-ERBα-deficiency. Ectopic overexpression in C2C12 cells demonstrated that REV-ERBß drives mitochondrial biogenesis and the expression of genes involved in fatty acid oxidation. Intriguingly, knock down of REV-ERBß in C2C12 cultures also resulted in mitochondrial biogenesis and increased expression of genes involved in fatty acid ß-oxidation. To determine whether these effects occurred in vivo, we examined REV-ERBß-deficient mice and observed a similar increase in expression of genes involved in mitochondrial biogenesis and fatty acid ß-oxidation. Consistent with these results, REV-ERBß-deficient mice exhibited an altered metabolic phenotype compared to wild-type littermate controls when measured by indirect calorimetry. This likely compensated for the increased food consumption that occurred, possibly aiding in the maintenance of their weight over time. Since feeding behaviors are a direct circadian output, this study suggests that REV-ERBß may have more subtle effects on circadian behaviors than originally identified. Furthermore, these data implicate REV-ERBß in the control of skeletal muscle metabolism and energy expenditure and suggest that development of REV-ERBα versus REV-ERBß selective ligands may have therapeutic utility in the treatment of metabolic syndrome.

Fatty acid transport protein 1 is required for nonshivering thermogenesis in brown adipose tissue.

Nonshivering thermogenesis in brown adipose tissue (BAT) generates heat through the uncoupling of mitochondrial beta-oxidation from ATP production. The principal energy source for this process is fatty acids that are either synthesized de novo in BAT or are imported from circulation. How uptake of fatty acids is mediated and regulated has remained unclear. Here, we show that fatty acid transport protein (FATP)1 is expressed on the plasma membrane of BAT and is upregulated in response to cold stimuli, concomitant with an increase in the rate of fatty acid uptake. In FATP1-null animals, basal fatty acid uptake is reduced and remains unchanged following cold exposure. As a consequence, FATP1 knockout (KO) animals display smaller lipid droplets in BAT and fail to defend their core body temperature at 4 degrees C, despite elevated serum free fatty acid levels. Similarly, FATP1 is expressed by the BAT-derived cell line HIB-1B upon differentiation, and both fatty acid uptake and FATP1 protein levels are rapidly elevated following isoproterenol stimulation. Stimulation of fatty uptake by isoproterenol required both protein kinase A and mitogen-activated kinase signaling and is completely dependent on FATP1 expression, as small-hairpin RNA-mediated knock down of FATP1 abrogated the effect.

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis.

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. In cancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach.

Dependence of brown adipose tissue function on CD36-mediated coenzyme Q uptake.

Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells synthesize CoQ mostly endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function are not well understood. Here, we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective nonshivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis.

Pharmacological targeting of the mammalian clock regulates sleep architecture and emotional behaviour.

Synthetic drug-like molecules that directly modulate the activity of key clock proteins offer the potential to directly modulate the endogenous circadian rhythm and treat diseases associated with clock dysfunction. Here we demonstrate that synthetic ligands targeting a key component of the mammalian clock, the nuclear receptors REV-ERBα and ß, regulate sleep architecture and emotional behaviour in mice. REV-ERB agonists induce wakefulness and reduce REM and slow-wave sleep. Interestingly, REV-ERB agonists also reduce anxiety-like behaviour. These data are consistent with increased anxiety-like behaviour of REV-ERBß-null mice, in which REV-ERB agonists have no effect. These results indicate that pharmacological targeting of REV-ERB may lead to the development of novel therapeutics to treat sleep disorders and anxiety.

PAZ6 cells constitute a representative model for human brown pre-adipocytes.

The role of brown adipose tissue (BAT) in human metabolism and its potential as an anti-obesity target organ have recently received much renewed attention. Following radiological detection of substantial amounts of BAT in adults by several independent research groups, an increasing number of studies are now dedicated to uncover BAT's genetic, developmental, and environmental determinants. In contrast to murine BAT, human BAT is not present as a single major fat depot in a well-defined location. The distribution of BAT in several areas in the body significantly limits its availability to research. A human brown adipocyte cell line is therefore critical in broadening the options available to researchers in the field. The human BAT-cell line PAZ6 was created to address such a need and has been well characterized by several research groups around the world. In the present review, we discuss their findings and propose potential applications of the PAZ6 cells in addressing the relevant questions in the BAT field, namely for future use in therapeutic applications.

Real-time noninvasive imaging of fatty acid uptake in vivo.

Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin sensitivity. To enable noninvasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models.

Silencing of hepatic fatty acid transporter protein 5 in vivo reverses diet-induced non-alcoholic fatty liver disease and improves hyperglycemia.

Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition, we utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic fatty acid transport protein 5 in vivo prior to or after establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrate here the ability to achieve specific, non-toxic, and persistent knockdown of fatty acid transport protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of fatty acid transport protein 5 was also able to reverse already established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. Thus, continued activity of hepatic fatty acid transport protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease.

Cancer cachexia modifies the zonal distribution of lipid metabolism-related proteins in rat liver.

Cancer cachexia is a syndrome that causes profound metabolic disruption. Lipid metabolism in the liver is markedly affected. We investigated the effect of cachexia upon liver-acinus lipid-metabolism zonation in Walker 245 carcinosarcoma-bearing rats (TB). The expression of protein (by Western blotting) and mRNA (by semi-quantitative polymerase chain reaction) of the enzymes of the carnitine palmitoyltransferase system (CPT I and CPT II) and of liver fatty-acid-binding protein (L-FABP) was studied. Although no changes were found for these parameters, the maximal activities (by radioassay) of CPT I and II were reduced (P<0.05) in TB compared with controls. CPT II activity in the perivenous (PV) region was higher in TB compared with controls. The distribution of CPT II and L-FABP (by immunohistochemistry) within the acinus was modified by cachexia: whereas CPT II positivity was restricted to the PV zone, L-FABP labelling shifted from periportal (control) to perivenous (TB) zone. These changes in metabolic zonation, together with decreased CPT II activity, may contribute to the aggravation of cachexia.