Phosphoglycolate phosphatase homologs act as glycerol-3-phosphate phosphatase to control stress and healthspan in C. elegans.

Metabolic stress due to nutrient excess and lipid accumulation is at the root of many age-associated disorders and the identification of therapeutic targets that mimic the beneficial effects of calorie restriction has clinical importance. Here, using C. elegans as a model organism, we study the roles of a recently discovered enzyme at the heart of metabolism in mammalian cells, glycerol-3-phosphate phosphatase (G3PP) (gene name Pgp) that hydrolyzes glucose-derived glycerol-3-phosphate to glycerol. We identify three Pgp homologues in C. elegans (pgph) and demonstrate in vivo that their protein products have G3PP activity, essential for glycerol synthesis. We demonstrate that PGPH/G3PP regulates the adaptation to various stresses, in particular hyperosmolarity and glucotoxicity. Enhanced G3PP activity reduces fat accumulation, promotes healthy aging and acts as a calorie restriction mimetic at normal food intake without altering fertility. Thus, PGP/G3PP can be considered as a target for age-related metabolic disorders.

Adipose ABHD6 regulates tolerance to cold and thermogenic programs.

Enhanced energy expenditure in brown (BAT) and white adipose tissues (WAT) can be therapeutic against metabolic diseases. We examined the thermogenic role of adipose α/ß-hydrolase domain 6 (ABHD6), which hydrolyzes monoacylglycerol (MAG), by employing adipose-specific ABHD6-KO mice. Control and KO mice showed similar phenotypes at room temperature and thermoneutral conditions. However, KO mice were resistant to hypothermia, which can be accounted for by the simultaneously increased lipolysis and lipogenesis of the thermogenic glycerolipid/free fatty acid (GL/FFA) cycle in visceral fat, despite unaltered uncoupling protein 1 expression. Upon cold stress, nuclear 2-MAG levels increased in visceral WAT of the KO mice. Evidence is provided that 2-MAG causes activation of PPARα in white adipocytes, leading to elevated expression and activity of GL/FFA cycle enzymes. In the ABHD6-ablated BAT, glucose and oxidative metabolism were elevated upon cold induction, without changes in GL/FFA cycle and lipid turnover. Moreover, response to in vivo ß3-adrenergic stimulation was comparable between KO and control mice. Our data reveal a MAG/PPARα/GL/FFA cycling metabolic signaling network in visceral adipose tissue, which contributes to cold tolerance, and that adipose ABHD6 is a negative modulator of adaptive thermogenesis.

α/ß-Hydrolase Domain 6 Deletion Induces Adipose Browning and Prevents Obesity and Type 2 Diabetes.

Suppression of α/ß-domain hydrolase-6 (ABHD6), a monoacylglycerol (MAG) hydrolase, promotes glucose-stimulated insulin secretion by pancreatic ß cells. We report here that high-fat-diet-fed ABHD6-KO mice show modestly reduced food intake, decreased body weight gain and glycemia, improved glucose tolerance and insulin sensitivity, and enhanced locomotor activity. ABHD6-KO mice also show increased energy expenditure, cold-induced thermogenesis, brown adipose UCP1 expression, fatty acid oxidation, and white adipose browning. Adipose browning and cold-induced thermogenesis are replicated by the ABHD6 inhibitor WWL70 and by antisense oligonucleotides targeting ABHD6. Our evidence suggests that one mechanism by which the lipolysis derived 1-MAG signals intrinsic and cell-autonomous adipose browning is via PPARα and PPARγ activation, and that ABHD6 regulates adipose browning by controlling signal competent 1-MAG levels. Thus, ABHD6 regulates energy homeostasis, brown adipose function, and white adipose browning and is a potential therapeutic target for obesity and type 2 diabetes.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/ß-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

In vivo voltage-sensitive dye imaging in adult mice reveals that somatosensory maps lost to stroke are replaced over weeks by new structural and functional circuits with prolonged modes of activation within both the peri-infarct zone and distant sites.

After brain damage such as stroke, topographically organized sensory and motor cortical representations remap onto adjacent surviving tissues. It is conceivable that cortical remapping is accomplished by changes in the temporal precision of sensory processing and regional connectivity in the cortex. To understand how the adult cortex remaps and processes sensory signals during stroke recovery, we performed in vivo imaging of sensory-evoked changes in membrane potential, as well as multiphoton imaging of dendrite structure and tract tracing. In control mice, forelimb stimulation evoked a brief depolarization in forelimb cortex that quickly propagated to, and dissipated within, adjacent motor/hindlimb areas (<100 ms). One week after forelimb cortex stroke, the cortex was virtually unresponsive to tactile forelimb stimulation. After 8 weeks recovery, forelimb-evoked depolarizations reemerged with a characteristic pattern in which responses began within surviving portions of forelimb cortex (<20 ms after stimulation) and then spread horizontally into neighboring peri-infarct motor/hindlimb areas in which depolarization persisted 300-400% longer than controls. These uncharacteristically prolonged responses were not limited to the remapped peri-infarct zone and included distant posteromedial retrosplenial cortex, millimeters from the stroke. Structurally, the remapped peri-infarct area selectively exhibited high levels of dendritic spine turnover, shared more connections with retrosplenial cortex and striatum, and lost inputs from lateral somatosensory cortical regions. Our findings demonstrate that sensory remapping during stroke recovery is accompanied by the development of prolonged sensory responses and new structural circuits in both the peri-infarct zone as well as more distant sites.

Dopamine activates Nrf2-regulated neuroprotective pathways in astrocytes and meningeal cells.

The transcription factor Nrf2 controls inducible expression of multiple antioxidant/detoxification genes. We previously found that Nrf2-/- mice have increased sensitivity to in vivo mitochondrial stress and ischemia. Although Nrf2 regulated these forms of neuronal toxicity, it was unclear which injury-triggered signal(s) led to Nrf2 activation in vivo. In this study, we use primary cultures to test the hypothesis that excessive dopamine release can act as an endogenous Nrf2-inducing signal. We cultured two cell types that show increased Nrf2 activity during ischemia in vivo, astrocytes and meningeal cells. Cultures were infected with an adenovirus reporter of Nrf2 transcriptional activity. Dopamine-induced Nrf2 activity in both cell types by generating oxidative stressors, H2O2 and dopamine-quinones. Nrf2 activation in meningeal cells was significantly higher than astrocytes. The effect of dopamine was blocked by antioxidants, and by over-expression of either dominant-negative Nrf2 or Keap1. Nrf2 induction was specific to oxidative stress caused by catecholaminergic neurotransmitters as epinephrine also induced Nrf2, but the monoamine serotonin had no significant effect. These in vitro results suggest Nrf2 activity in astrocytes and meningeal cells link the neurotoxic actions of dopamine to neuroprotective pathways that may potentially modulate ischemic injury and neurodegeneration.

Cystine/glutamate exchange modulates glutathione supply for neuroprotection from oxidative stress and cell proliferation.

The cystine/glutamate exchanger (xCT) provides intracellular cyst(e)ine for production of glutathione, a major cellular antioxidant. Using xCT overexpression and underexpression, we present evidence that xCT-dependent glutathione production modulates both neuroprotection from oxidative stress and cell proliferation. In embryonic and adult rat brain, xCT protein was enriched at the CSF-brain barrier (i.e., meninges) and also expressed in the cortex, hippocampus, striatum, and cerebellum. To examine the neuroprotective role of xCT, various non-neuronal cell types (astrocytes, meningeal cells, and peripheral fibroblasts) were cocultured with immature cortical neurons and exposed to oxidative glutamate toxicity, a model involving glutathione depletion. Cultured meningeal cells, which naturally maintain high xCT expression, were more neuroprotective than astrocytes. Selective xCT overexpression in astrocytes was sufficient to enhance glutathione synthesis/release and confer potent glutathione-dependent neuroprotection from oxidative stress. Moreover, normally nonprotective fibroblasts could be re-engineered to be neuroprotective with ectopic xCT overexpression indicating that xCT is a key step in the pathway to glutathione synthesis. Conversely, astrocytes and meningeal cells derived from sut/sut mice (xCT loss-of-function mutants) showed greatly reduced proliferation in culture attributable to increased oxidative stress and thiol deficiency, because growth could be rescued by the thiol-donor beta-mercaptoethanol. Strikingly, sut/sut mice developed brain atrophy by early adulthood, exhibiting ventricular enlargement, thinning of the cortex, and shrinkage of the striatum. Our results indicate that xCT can provide neuroprotection by enhancing glutathione export from non-neuronal cells such as astrocytes and meningeal cells. Furthermore, xCT is critical for cell proliferation during development in vitro and possibly in vivo.

Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface.

Glutathione is the major cellular thiol present in mammalian cells and is critical for maintenance of redox homeostasis. However, current assay systems for glutathione lack application to intact animal tissues. To map the levels of glutathione in intact brain with cellular resolution (acute tissue slices and live animals), we have used two-photon imaging of monochlorobimane fluorescence, a selective enzyme-mediated marker for reduced glutathione. Previously, in vitro experiments using purified components and cultured glial cells attributed cellular monochlorobimane fluorescence to a glutathione S-transferase-dependent reaction with GSH. Our results indicate that cells at the cerebrospinal fluid or blood-brain interface, such as lateral ventricle ependymal cells (2.73 +/- 0.56 mm; glutathione), meningeal cells (1.45 +/- 0.09 mm), and astroglia (0.91 +/- 0.08 mm), contain high levels of glutathione. In comparison, layer II cortical neurons contained 20% (0.21 +/- 0.02 mm) the glutathione content of nearby astrocytes. Neuronal glutathione labeling increased 250% by the addition of the cell-permeable glutathione precursor N-acetylcysteine indicating that the monochlorobimane level or glutathione S-transferase activity within neurons was not limiting. Regional mapping showed that glutathione was highest in cells lining the lateral ventricles, specifically ependymal cells and the subventricular zone, suggesting a possible function for glutathione in oxidant homeostasis of developing neuronal progenitors. Consistently, developing neurons in the subgranular zone of dentate gyrus contained 3-fold more glutathione than older neurons found in the neighboring granular layer. In conclusion, mapping of glutathione levels in intact brain demonstrates a unique role for enhanced redox potential in developing neurons and cells at the cerebrospinal fluid and blood-brain interface.

Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo.

NF-E2 related factor (Nrf2) controls a pleiotropic cellular defense, where multiple antioxidant/detoxification pathways are up-regulated in unison. Although small molecule inducers of Nrf2 activity have been reported to protect neurons in vitro, whether similar pathways can be accessed in vivo is not known. We have investigated whether in vivo toxicity of the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP) can be attenuated by constitutive and inducible Nrf2 activity. The absence of Nrf2 function in Nrf2(-/-) mice resulted in 3-NP hypersensitivity that became apparent with time and increasing dose, causing motor deficits and striatal lesions on a more rapid time scale than identically treated Nrf2(+/+) and Nrf2(+/-) controls. Striatal succinate dehydrogenase activity, the target of 3-NP, was inhibited to the same extent in all genotypes by a single acute dose of 3-NP, suggesting that brain concentrations of 3-NP were similar. Dietary supplementation with the Nrf2 inducer tert-butylhydroquinone attenuated 3-NP toxicity in Nrf2(+/-) mice, but not Nrf2(-/-), confirming the Nrf2-specific action of the inducer in vivo. Increased Nrf2 activity alone was sufficient to protect animals from 3-NP toxicity because intrastriatal adenovirus-mediated Nrf2 overexpression significantly reduced lesion size compared with green fluorescent protein overexpressing controls. In cultured astrocytes, 3-NP was found to increase Nrf2 activity leading to antioxidant response element-dependent gene expression providing a potential mechanism for the increased sensitivity of Nrf2(-/-) animals to 3-NP toxicity in vivo. We conclude that Nrf2 may underlie a feedback system limiting oxidative load during chronic metabolic stress.

Coordinate regulation of glutathione metabolism in astrocytes by Nrf2.

The tripeptide glutathione (GSH) represents the major brain thiol and is essential for prevention of oxidative stress. Using monochlorobimane to label intracellular GSH in a glutathione S-transferase catalyzed reaction we have examined the kinetics of GSH metabolism including its rate of conjugation, total GSH content, synthesis, and efflux in astrocyte cultures under basal conditions and after induction of antioxidant response element (ARE)-mediated gene expression by the transcription factor Nrf2. In the presence of a cerebral spinal fluid-like salt solution astrocytes could not synthesize detectable levels of GSH. Addition of GSH precursors, cystine, glutamate, and glycine, rapidly restored GSH synthesis. Astrocytes were able to use either glutamate or glutamine as precursors equally for GSH synthesis. Using the small molecule chemical inducer tert-butylhydroqunione (tBHQ) we report that induction of ARE-mediated gene expression is associated with a coordinated increase in GSH content and synthesis rate with little effect on the rate of GSH conjugation or efflux. Consistent with the effect of the inducer, adenovirus-mediated overexpression of the transcription factor Nrf2 that mediates tBHQ's effects also increased GSH content, confirming that GSH metabolism can be regulated by the Nrf2 pathway.

Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress.

Astrocytes have a higher antioxidant potential in comparison to neurons. Pathways associated with this selective advantage include the transcriptional regulation of antioxidant enzymes via the action of the Cap'n'Collar transcription factor Nrf2 at the antioxidant response element (ARE). Here we show that Nrf2 overexpression can reengineer neurons to express this glial pathway and enhance antioxidant gene expression. However, Nrf2-mediated protection from oxidative stress is conferred primarily by glia in mixed cultures. The antioxidant properties of Nrf2-overexpressing glia are more pronounced than those of neurons, and a relatively small number of these glia (< 1% of total cell number added) could protect fully cocultured naive neurons from oxidative glutamate toxicity associated with glutathione (GSH) depletion. Microarray and biochemical analyses indicate a coordinated upregulation of enzymes involved in GSH biosynthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase, and GSH synthase), use (glutathione S-transferase and glutathione reductase), and export (multidrug resistance protein 1) with Nrf2 overexpression, leading to an increase in both media and intracellular GSH. Selective inhibition of glial GSH synthesis and the supplementation of media GSH indicated that an Nrf2-dependent increase in glial GSH synthesis was both necessary and sufficient for the protection of neurons, respectively. Neuroprotection was not limited to overexpression of Nrf2, because activation of endogenous glial Nrf2 by the small molecule ARE inducer, tert-butylhydroquinone, also protected against oxidative glutamate toxicity.