Circumventricular organ-hypothalamic circuit endoplasmic reticulum stress drives hepatic steatosis during obesity.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), characterized by excess liver triglyceride accumulation (hepatic steatosis), leads to an increased risk for cardiometabolic diseases and obesity-related mortality. Emerging evidence points to endoplasmic reticulum (ER) stress in the central nervous system as critical in NAFLD pathogenesis. Here, we tested the contribution of ER stress in a circumventricular organ-hypothalamic circuit in NAFLD development during obesity. METHODS: C57BL/6J male mice were fed a high-fat diet (HFD) or normal chow. A combination of histological, viral tracing, intersectional viral targeting, and in vivo integrative physiological approaches were used to examine the role of ER stress in subfornical organ to hypothalamic paraventricular nucleus projecting neurons (SFO➔PVN) in NAFLD during diet-induced obesity. RESULTS: Immunohistochemical analysis revealed marked unfolded protein response activation in the SFO, particularly in excitatory SFO➔PVN neurons of HFD-fed animals. Moreover, intersectional viral inhibition of ER stress in SFO➔PVN neurons resulted in a reduction in hepatomegaly, hepatic steatosis, and a blunted increase in body weight gain during diet-induced obesity, independent of changes in food intake, substrate partitioning, energy expenditure, and ambulatory activity. CONCLUSIONS: These results indicate that ER stress in an SFO➔PVN neural circuit contributes to hepatic steatosis during obesity.

A forebrain-hypothalamic ER stress driven circuit mediates hepatic steatosis during obesity.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) affects 1 in 3 adults and contributes to advanced liver injury and cardiometabolic disease. While recent evidence points to involvement of the brain in NAFLD, the downstream neural circuits and neuronal molecular mechanisms involved in this response, remain unclear. Here, we investigated the role of a unique forebrain-hypothalamic circuit in NAFLD. METHODS: Chemogenetic activation and inhibition of circumventricular subfornical organ (SFO) neurons that project to the paraventricular nucleus of the hypothalamus (PVN; SFO→PVN) in mice were used to study the role of SFO→PVN signaling in NAFLD. Novel scanning electron microscopy techniques, histological approaches, molecular biology techniques, and viral methodologies were further used to delineate the role of endoplasmic reticulum (ER) stress within this circuit in driving NAFLD. RESULTS: In lean animals, acute chemogenetic activation of SFO→PVN neurons was sufficient to cause hepatic steatosis in a liver sympathetic nerve dependent manner. Conversely, inhibition of this forebrain-hypothalamic circuit rescued obesity-associated NAFLD. Furthermore, dietary NAFLD is associated with marked ER ultrastructural alterations and ER stress in the PVN, which was blunted following reductions in excitatory signaling from the SFO. Finally, selective inhibition of PVN ER stress reduced hepatic steatosis during obesity. CONCLUSIONS: Collectively, these findings characterize a previously unrecognized forebrain-hypothalamic-ER stress circuit that is involved in hepatic steatosis, which may point to future therapeutic strategies for NAFLD.

Bridging Energy Need and Feeding Behavior: The Impact of eIF2α Phosphorylation in AgRP Neurons.

Eukaryotic translation initiation factor 2α (eIF2α) is a key mediator of the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR). In mammals, eIF2α is phosphorylated by overnutrition-induced ER stress and is related to the development of obesity. Here, we studied the function of phosphorylated eIF2α (p-eIF2α) in agouti-related peptide (AgRP) neurons using a mouse model (AgRPeIF2αA/A) with an AgRP neuron-specific substitution from Ser 51 to Ala in eIF2α, which impairs eIF2α phosphorylation in AgRP neurons. These AgRPeIF2αA/A mice had decreases in starvation-induced AgRP neuronal activity and food intake and an increased responsiveness to leptin. Intriguingly, impairment of eIF2α phosphorylation produced decreases in the starvation-induced expression of UPR and autophagy genes in AgRP neurons. Collectively, these findings suggest that eIF2α phosphorylation regulates AgRP neuronal activity by affecting intracellular responses such as the UPR and autophagy during starvation, thereby participating in the homeostatic control of whole-body energy metabolism. ARTICLE HIGHLIGHTS: This study examines the impact of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, triggered by an energy deficit, on hypothalamic AgRP neurons and its subsequent influence on whole-body energy homeostasis. Impaired eIF2α phosphorylation diminishes the unfolded protein response and autophagy, both of which are crucial for energy deficit-induced activation of AgRP neurons. This study highlights the significance of eIF2α phosphorylation as a cellular marker indicating the availability of energy in AgRP neurons and as a molecular switch that regulates homeostatic feeding behavior.

Transcriptional Regulatory Role of NELL2 in Preproenkephalin Gene Expression.

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-likelike 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

Spexin Regulates Hypothalamic Leptin Action on Feeding Behavior.

Spexin (SPX) is a recently identified neuropeptide that is believed to play an important role in the regulation of energy homeostasis. Here, we describe a mediating function of SPX in hypothalamic leptin action. Intracerebroventricular (icv) SPX administration induced a decrease in food intake and body weight gain. SPX was found to be expressed in cells expressing leptin receptor ObRb in the mouse hypothalamus. In line with this finding, icv leptin injection increased SPX mRNA in the ObRb-positive cells of the hypothalamus, which was blocked by treatment with a STAT3 inhibitor. Leptin also increased STAT3 binding to the SPX promoter, as measured by chromatin immunoprecipitation assays. In vivo blockade of hypothalamic SPX biosynthesis with an antisense oligodeoxynucleotide (AS ODN) resulted in a diminished leptin effect on food intake and body weight. AS ODN reversed leptin's effect on the proopiomelanocortin (POMC) mRNA expression and, moreover, decreased leptin-induced STAT3 binding to the POMC promoter sequence. These results suggest that SPX is involved in leptin's action on POMC gene expression in the hypothalamus and impacts the anorexigenic effects of leptin.

NELL2 Function in Axon Development of Hippocampal Neurons.

Neurons have multiple dendrites and single axon. This neuronal polarity is gradually established during early processes of neuronal differentiation: generation of multiple neurites (stages 1-2); differentiation (stage 3) and maturation (stages 4-5) of an axon and dendrites. In this study, we demonstrated that the neuron-specific n-glycosylated protein NELL2 is important for neuronal polarization and axon growth using cultured rat embryonic hippocampal neurons. Endogenous NELL2 expression was gradually increased in parallel with the progression of developmental stages of hippocampal neurons, and overexpression of NELL2 stimulated neuronal polarization and axon growth. In line with these results, knockdown of NELL2 expression resulted in deterioration of neuronal development, including inhibition of neuronal development progression, decreased axon growth and increased axon branching. Inhibitor against extracellular signal-regulated kinase (ERK) dramatically inhibited NELL2-induced progression of neuronal development and axon growth. These results suggest that NELL2 is an important regulator for the morphological development for neuronal polarization and axon growth.

Brain-specific chemokine FAM19A5 induces hypothalamic inflammation.

The cytokine-like protein FAM19A5 is highly expressed in the brain, but little is known about its functions there. Here, we found that FAM19A5 was expressed in mouse hypothalamic cells expressing proopiomelanocortin (POMC) and neuropeptide Y (NPY)/agouti-related peptide (AgRP), and in the microglia. Tumor necrosis factor-α (TNF-α), which induces inflammatory sickness responses, greatly increased hypothalamic expression of FAM19A5. Knockdown of FAM19A5 expression resulted in decreased TNF-α-induced anorexia, body weight loss and TNF-α-induced expression of inflammatory factors. In contrast, intracerebroventricular administration of FAM19A5 induced anorexia, body weight loss and hyperthermia, together with increased expression of inflammatory factors. FAM19A5 injection also induced increases in c-fos activation and POMC mRNA level in hypothalamic POMC neurons. Together, these results suggest that FAM19A5 plays an important role in hypothalamic inflammatory responses.

Tonicity-responsive enhancer binding protein (TonEBP) regulates TNF-α-induced hypothalamic inflammation.

Tonicity-responsive enhancer binding protein (TonEBP) is a widely expressed transcription factor and is important in the regulation of inflammatory cytokines. Here, we have identified TonEBP expression in the hypothalamus, which is particularly high in proopiomelanocortin (POMC) neurons. TonEBP overexpression stimulates POMC transcription, and TonEBP haploinsufficiency in TonEBP (+/-) mice results in a decrease in hypothalamic POMC expression. TonEBP (+/-) mice show reduced sickness responses, which include anorexia and hyperthermia, that are initially induced by tumor necrosis factor (TNF)-α. TonEBP (+/-) mice also show lower levels of TNF-α-induced hypothalamic expression of POMC and pro-inflammatory cytokines. These results suggest that TonEBP is an important molecular regulator in the development of inflammatory sickness responses through the control of POMC and pro-inflammatory cytokine expression in the hypothalamus.

Role of thyroid transcription factor-1 in transcriptional regulation of heme oxygenase-1.

Here, we report thyroid transcription factor 1 (TTF-1) as an important transcription factor for the expression of heme oxygenase-1 (HO-1). HO-1 is a well-known cytoprotective enzyme against inflammation. We observed that HO-1 co-expressed with TTF-1 in mouse hypothalamic cells. Results from luciferase and chromatin immunoprecipitation assays revealed that TTF-1 directly activated HO-1 transcription by binding to binding domains in the 5'-flanking region of the HO-1 gene. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), induced nuclear translocation of TTF-1 and increased binding affinity of TTF-1 to its binding sites on the HO-1 gene. HO-1 mRNA increased with TTF-1 overexpression but decreased with RNA interference of TTF-1 expression in rat astroglial C6 cells. Together with results showing involvement of TTF-1 in the TNF-α-induced increase in interleukin 1 beta and monocyte chemotactic protein 1 production, this study suggests that TTF-1 plays an important role in the mouse hypothalamus TNF-α-induced inflammatory response for regulating HO-1 gene expression.

A Role of Central NELL2 in the Regulation of Feeding Behavior in Rats.

A brain-enriched secreting signal peptide, NELL2, has been suggested to play multiple roles in the development, survival, and activity of neurons in mammal. We investigated here a possible involvement of central NELL2 in regulating feeding behavior and metabolism. In situ hybridization and an im-munohistochemical approach were used to determine expression of NELL2 as well as its colocalization with proopiomelanocortin (POMC) and neuropeptide Y (NPY) in the rat hypothalamus. To investigate the effect of NELL2 on feeding behavior, 2 nmole of antisense NELL2 oligodeoxynucleotide was administered into the lateral ventricle of adult male rat brains for 6 consecutive days, and changes in daily body weight, food, and water intake were monitored. Metabolic state-dependent NELL2 expression in the hypothalamus was tested in vivo using a fasting model. NELL2 was noticeably expressed in the hypothalamic nuclei controlling feeding behavior. Furthermore, all arcuatic POMC and NPY positive neurons produced NELL2. The NELL2 gene expression in the hypothalamus was up-regulated by fasting. However, NELL2 did not affect POMC and NPY gene expression in the hypothalamus. A blockade of NELL2 production in the hypothalamus led to a reduction in daily food intake, followed by a loss in body weight without a change in daily water intake in normal diet condition. NELL2 did not affect short-term hunger dependent appetite behavior. Our data suggests that hypothalamic NELL2 is associated with appetite behavior, and thus central NELL2 could be a new therapeutic target for obesity.

NELL2 function in the protection of cells against endoplasmic reticulum stress.

Continuous intra- and extracellular stresses induce disorder of Ca(2+) homeostasis and accumulation of unfolded protein in the endoplasmic reticulum (ER), which results in ER stress. Severe long-term ER stress triggers apoptosis signaling pathways, resulting in cell death. Neural epidermal growth factor-like like protein 2 (NELL2) has been reported to be important in protection of cells from cell death-inducing environments. In this study, we investigated the cytoprotective effect of NELL2 in the context of ER stress induced by thapsigargin, a strong ER stress inducer, in Cos7 cells. Overexpression of NELL2 prevented ER stress-mediated apoptosis by decreasing expression of ER stress-induced C/EBP homologous protein (CHOP) and increasing ER chaperones. In this context, expression of anti-apoptotic Bcl-xL was increased by NELL2, whereas NELL2 decreased expression of pro-apoptotic proteins, such as cleaved caspases 3 and 7. This anti-apoptotic effect of NELL2 is likely mediated by extracellular signal-regulated kinase (ERK) signaling, because its inhibitor, U0126, inhibited effects of NELL2 on the expression of anti- and pro-apoptotic proteins and on the protection from ER stress-induced cell death.

Neural epidermal growth factor-like like protein 2 (NELL2) promotes aggregation of embryonic carcinoma P19 cells by inducing N-cadherin expression.

NELL2 was first identified as a mammalian homolog of chick NEL (Neural EGF-like) protein. It is almost exclusively expressed in neurons of the rat brain and has been suggested to play a role in neural differentiation. However, there is still no clear evidence for the detailed function of NELL2 in the differentiation of neurons. In this study, we identified NELL2 function during neural differentiation of mouse embryonic carcinoma P19 cells. Endogenous expression of NELL2 in the P19 cells increased in parallel with the neuronal differentiation induced by retinoic acid (RA). We found that the mouse NELL2 promoter contains RA response elements (RAREs) and that treatment with RA increased NELL2 promoter activity. Transfection of P19 cells with NELL2 expression vectors induced a dramatic increase in cell aggregation, resulting in the facilitation of neural differentiation. Moreover, NELL2 significantly increased N-cadherin expression in the P19 cell. These data suggest that NELL2 plays an important role in the regulation of neuronal differentiation via control of N-cadherin expression and cell aggregation.

Regulation of the female rat estrous cycle by a neural cell-specific epidermal growth factor-like repeat domain containing protein, NELL2.

NELL2, a protein containing epidermal growth factor-like repeat domains, is predominantly expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. Previously we found that NELL2 is involved in the onset of female puberty by regulating the release of gonadotropin-releasing hormone (GnRH), and in normal male sexual behavior by controlling the development of the sexually dimorphic nucleus of the preoptic area (POA). In this study we investigated the effect of NELL2 on the female rat estrous cycle. NELL2 expression in the POA was highest during the proestrous phase. NELL2 mRNA levels in the POA were increased by estrogen treatment in ovariectomized female rats. Blocking NELL2 synthesis in the female rat hypothalamus decreased the expression of kisspeptin 1, an important regulator of the GnRH neuronal apparatus, and resulted in disruption of the estrous cycle at the diestrous phase. These results indicate that NELL2 is involved in the maintenance of the normal female reproductive cycle in mammals.

Region- and neuronal phenotype-specific expression of NELL2 in the adult rat brain.

NELL2, a neural tissue-enriched protein, is produced in the embryo, and postembryonically in the mammalian brain, with a broad distribution. Although its synthesis is required for neuronal differentiation in chicks, not much is known about its function in the adult mammalian brain. We investigated the distribution of NELL2 in various regions of the adult rat brain to study its potential functions in brain physiology. Consistent with previous reports, NELL2-immunoreactivity (ir) was found in the cytoplasm of neurons, but not in glial fibrillary acidic protein (GFAP)-positive glial cells. The highest levels of NELL2 were detected in the hippocampus and the cerebellum. Interestingly, in the cerebellar cortex NELL2 was observed only in the GABAergic Purkinje cells not in the excitatory granular cells. In contrast, it was found mainly in the hippocampal dentate gyrus and pyramidal cell layer that contains mainly glutamatergic neurons. In the dentate gyrus, NELL2 was not detected in the GFAP-positive neural precursor cells, but was generally present in mature neurons of the subgranular zone, suggesting a role in this region restricted to mature neurons.