Time-Restricted Feeding Attenuates Metabolic Dysfunction-Associated Steatohepatitis and Hepatocellular Carcinoma in Obese Male Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) has surpassed the hepatitis B virus and hepatitis C virus as the leading cause of chronic liver disease in most parts of the Western world. MASLD (formerly known as NAFLD) encompasses both simple steatosis and more aggressive metabolic dysfunction-associated steatohepatitis (MASH), which is accompanied by inflammation, fibrosis, and cirrhosis, and ultimately can lead to hepatocellular carcinoma (HCC). There are currently very few approved therapies for MASH. Weight loss strategies such as caloric restriction can ameliorate the harmful metabolic effect of MASH and inhibit HCC; however, it is difficult to implement and maintain in daily life, especially in individuals diagnosed with HCC. In this study, we tested a time-restricted feeding (TRF) nutritional intervention in mouse models of MASH and HCC. We show that TRF abrogated metabolic dysregulation induced by a Western diet without any calorie restriction or weight loss. TRF improved insulin sensitivity and reduced hyperinsulinemia, liver steatosis, inflammation, and fibrosis. Importantly, TRF inhibited liver tumors in two mouse models of obesity-driven HCC. Our data suggest that TRF is likely to be effective in abrogating MASH and HCC and warrant further studies of time-restricted eating in humans with MASH who are at higher risk of developing HCC.

TM7SF3 controls TEAD1 splicing to prevent MASH-induced liver fibrosis.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Restoring SRSF3 in Kupffer cells attenuates obesity-related insulin resistance.

BACKGROUND AND AIMS: In obesity, depletion of KCs expressing CRIg (complement receptor of the Ig superfamily) leads to microbial DNA accumulation, which subsequently triggers tissue inflammation and insulin resistance. However, the mechanism underlying obesity-mediated changes in KC complement immune functions is largely unknown. APPROACH AND RESULTS: Using KC-specific deactivated Cas9 transgenic mice treated with guide RNA, we assessed the effects of restoring CRIg or the serine/arginine-rich splicing factor 3 (SRSF3) abundance on KC functions and metabolic phenotypes in obese mice. The impacts of weight loss on KC responses were evaluated in a diet switch mouse model. The role of SRSF3 in regulating KC functions was also evaluated using KC-specific SRSF3 knockout mice. Here, we report that overexpression of CRIg in KCs of obese mice protects against bacterial DNA accumulation in metabolic tissues. Mechanistically, SRSF3 regulates CRIg expression, which is essential for maintaining the CRIg+ KC population. During obesity, SRSF3 expression decreases, but it is restored with weight loss through a diet switch, normalizing CRIg+ KCs. KC SRSF3 is also repressed in obese human livers. Lack of SRSF3 in KCs in lean and obese mice decreases their CRIg+ population, impairing metabolic parameters. During the diet switch, the benefits of weight loss are compromised due to SRSF3 deficiency. Conversely, SRSF3 overexpression in obese mice preserves CRIg+ KCs and improves metabolic responses. CONCLUSIONS: Restoring SRSF3 abundance in KCs offers a strategy against obesity-associated tissue inflammation and insulin resistance by preventing bacterial DNA accumulation.

Dysregulation of RNA splicing in early non-alcoholic fatty liver disease through hepatocellular carcinoma.

While changes in RNA splicing have been extensively studied in hepatocellular carcinoma (HCC), no studies have systematically investigated changes in RNA splicing during earlier liver disease. Mouse studies have shown that disruption of RNA splicing can trigger liver disease and we have shown that the splicing factor SRSF3 is decreased in the diseased human liver, so we profiled RNA splicing in liver samples from twenty-nine individuals with no-history of liver disease or varying degrees of non-alcoholic fatty liver disease (NAFLD). We compared our results with three publicly available transcriptome datasets that we re-analyzed for splicing events (SEs). We found many changes in SEs occurred during early liver disease, with fewer events occurring with the onset of inflammation and fibrosis. Many of these early SEs were enriched for SRSF3-dependent events and were associated with SRSF3 binding sites. Mapping the early and late changes to gene ontologies and pathways showed that the genes harboring these early SEs were involved in normal liver metabolism, whereas those harboring late SEs were involved in inflammation, fibrosis and proliferation. We compared the SEs with HCC data from the TCGA and observed that many of these early disease SEs are found in HCC samples and, furthermore, are correlated with disease survival. Changes in splicing factor expression are also observed, which may be associated with distinct subsets of the SEs. The maintenance of these SEs through the multi-year oncogenic process suggests that they may be causative. Understanding the role of these splice variants in metabolic liver disease progression may shed light on the triggers of liver disease progression and the pathogenesis of HCC.

Loss of cAMP Signaling in CD11c Immune Cells Protects Against Diet-Induced Obesity.

In obesity, CD11c+ innate immune cells are recruited to adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. We found that ablation of Gnas, the gene that encodes Gαs, in CD11c expressing cells protects mice from obesity, glucose intolerance, and insulin resistance. Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and did not require adaptive immunity. Loss of cAMP signaling was associated with increased adipose tissue norepinephrine and cAMP signaling, and prevention of catecholamine resistance. The adipose tissue had reduced expression of catecholamine transport and degradation enzymes, suggesting that the elevated norepinephrine resulted from decreased catabolism. Collectively, our results identified an important role for cAMP signaling in CD11c+ innate immune cells in whole-body metabolism by controlling norepinephrine levels in white adipose tissue, modulating catecholamine-induced lipolysis and increasing thermogenesis, which, together, created a lean phenotype. ARTICLE HIGHLIGHTS: We undertook this study to understand how immune cells communicate with adipocytes, specifically, whether cAMP signaling in the immune cell and the adipocyte are connected. We identified a reciprocal interaction between CD11c+ innate immune cells and adipocytes in which high cAMP signaling in the immune cell compartment induces low cAMP signaling in adipocytes and vice versa. This interaction regulates lipolysis in adipocytes and inflammation in immune cells, resulting in either a lean, obesity-resistant, and insulin-sensitive phenotype, or an obese, insulin-resistant phenotype.

Treatment of Hepatocellular Carcinoma by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant.

PURPOSE: To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Mice with bilateral, subcutaneous RIL-175 cell-derived HCCs were randomized to 4 groups: (a) phosphate-buffered saline (control), (b) cryoablation only (Cryo), (c) CPMV-treated only (CPMV), and (d) cryoablation plus CPMV-treated (Cryo + CPMV) (N = 11-14 per group). Intratumoral CPMV was administered every 3 days for 4 doses, with cryoablation performed on the third day. Contralateral tumors were monitored. Tumor growth and systemic chemokine/cytokine levels were measured. A subset of tumors and spleens were harvested for immunohistochemistry (IHC) and flow cytometry. One- or 2-way analysis of variance was performed for statistical comparisons. A P value of <.05 was used as the threshold for statistical significance. RESULTS: At 2 weeks after treatment, the Cryo and CPMV groups, alone or combined, outperformed the control group in the treated tumor; however, the Cryo + CPMV group showed the strongest reduction and lowest variance (1.6-fold ± 0.9 vs 6.3-fold ± 0.5, P < .0001). For the untreated tumor, only Cryo + CPMV significantly reduced tumor growth compared with control (9.2-fold ± 0.9 vs 17.8-fold ± 2.1, P = .01). The Cryo + CPMV group exhibited a transient increase in interleukin-10 and persistently decreased CXCL1. Flow cytometry revealed natural killer cell enrichment in the untreated tumor and increased PD-1 expression in the spleen. Tumor-infiltrating lymphocytes increased in Cryo + CPMV-treated tumors by IHC. CONCLUSIONS: Cryoablation and intratumoral CPMV, alone or combined, demonstrated potent efficacy against treated HCC tumors; however, only cryoablation combined with CPMV slowed the growth of untreated tumors, consistent with an abscopal effect.

Long-term exposure to house dust mites accelerates lung cancer development in mice.

BACKGROUND: Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1ß (IL-1ß), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1ß signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1ß, and CCL2 neutralization, or ICS treatment. CONCLUSIONS: Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing.

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans -membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3'end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

The ECRG4 cleavage product augurin binds the endotoxin receptor and influences the innate immune response during otitis media.

Otitis media (OM), the most common disease of childhood, is typically characterized by bacterial infection of the middle ear (ME). Prominent features of OM include hyperplasia of the ME mucosa, which transforms from a monolayer of simple squamous epithelium with minimal stroma into a full-thickness respiratory epithelium in 2-3 days after infection. Analysis of the murine ME transcriptome during OM showed down-regulation of the tumor suppressor gene Ecrg4 that was temporally related to mucosal hyperplasia and identified stromal cells as the primary ECRG4 source. The reduction in Ecrg4 gene expression coincided with the cleavage of ECRG4 protein to release an extracellular fragment, augurin. The duration of mucosal hyperplasia during OM was greater in Ecrg4 -/- mice, the number of infiltrating macrophages was enhanced, and ME infection cleared more rapidly. ECRG4-null macrophages showed increased bacterial phagocytosis. Co-immunoprecipitation identified an association of augurin with TLR4, CD14 and MD2, the components of the lipopolysaccharide (LPS) receptor. The results suggest that full-length ECRG4 is a sentinel molecule that potentially inhibits growth of the ME stroma. Processing of ECRG4 protein during inflammation, coupled with a decline in Ecrg4 gene expression, also influences the behavior of cells that do not express the gene, limiting the production of growth factors by epithelial and endothelial cells, as well as the activity of macrophages.

Obesity, cancer risk, and time-restricted eating.

Obesity and the associated metabolic syndrome is considered a pandemic whose prevalence is steadily increasing in many countries worldwide. It is a complex, dynamic, and multifactorial disorder that presages the development of several metabolic, cardiovascular, and neurodegenerative diseases, and increases the risk of cancer. In patients with newly diagnosed cancer, obesity worsens prognosis, increasing the risk of recurrence and decreasing survival. The multiple negative effects of obesity on cancer outcomes are substantial, and of great clinical importance. Strategies for weight control have potential utility for both prevention efforts and enhancing cancer outcomes. Presently, time-restricted eating (TRE) is a popular dietary intervention that involves limiting the consumption of calories to a specific window of time without any proscribed caloric restriction or alteration in dietary composition. As such, TRE is a sustainable long-term behavioral modification, when compared to other dietary interventions, and has shown many health benefits in animals and humans. The preliminary data regarding the effects of time-restricted feeding on cancer development and growth in animal models are promising but studies in humans are lacking. Interestingly, several short-term randomized clinical trials of TRE have shown favorable effects to reduce cancer risk factors; however, long-term trials of TRE have yet to investigate reductions in cancer incidence or outcomes in the general population. Few studies have been conducted in cancer populations, but a number are underway to examine the effect of TRE on cancer biology and recurrence. Given the simplicity, feasibility, and favorable metabolic improvements elicited by TRE in obese men and women, TRE may be useful in obese cancer patients and cancer survivors; however, the clinical implementation of TRE in the cancer setting will require greater in-depth investigation.

Hepatocyte Deletion of IGF2 Prevents DNA Damage and Tumor Formation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Serine-arginine rich splicing factor 3 (SRSF3) plays a critical role in hepatocyte function and its loss in mice promotes chronic liver damage and leads to HCC. Hepatocyte-specific SRSF3 knockout mice (SKO mice) also overexpress insulin-like growth factor 2 (IGF2). In the present study, double deletion of Igf2 and Srsf3 (DKO mice) prevents hepatic fibrosis and inflammation, and completely prevents tumor formation, and is associated with decreased proliferation, apoptosis and DNA damage, and restored DNA repair enzyme expression. This is confirmed in vitro, where IGF2 treatment of HepG2 hepatoma cells decreases DNA repair enzyme expression and causes DNA damage. Tumors from the SKO mice also show mutational signatures consistent with homologous recombination and mismatch repair defects. Analysis of frozen human samples shows that SRSF3 protein is decreased sixfold in HCC compared to normal liver tissue but SRSF3 mRNA is increased. Looking at public TCGA data, HCC patients having high SRSF3 mRNA expression show poor survival, as do patients with alterations in known SRSF3-dependent splicing events. The results indicate that IGF2 overexpression in conjunction with reduced SRSF3 splicing activity could be a major cause of DNA damage and driver of liver cancer.

Catestatin induces glycogenesis by stimulating the phosphoinositide 3-kinase-AKT pathway.

AIM: Defects in hepatic glycogen synthesis contribute to post-prandial hyperglycaemia in type 2 diabetic patients. Chromogranin A (CgA) peptide Catestatin (CST: hCgA352-372 ) improves glucose tolerance in insulin-resistant mice. Here, we seek to determine whether CST induces hepatic glycogen synthesis. METHODS: We determined liver glycogen, glucose-6-phosphate (G6P), uridine diphosphate glucose (UDPG) and glycogen synthase (GYS2) activities; plasma insulin, glucagon, noradrenaline and adrenaline levels in wild-type (WT) as well as in CST knockout (CST-KO) mice; glycogen synthesis and glycogenolysis in primary hepatocytes. We also analysed phosphorylation signals of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-dependent kinase-1 (PDK-1), GYS2, glycogen synthase kinase-3ß (GSK-3ß), AKT (a kinase in AKR mouse that produces Thymoma)/PKB (protein kinase B) and mammalian/mechanistic target of rapamycin (mTOR) by immunoblotting. RESULTS: CST stimulated glycogen accumulation in fed and fasted liver and in primary hepatocytes. CST reduced plasma noradrenaline and adrenaline levels. CST also directly stimulated glycogenesis and inhibited noradrenaline and adrenaline-induced glycogenolysis in hepatocytes. In addition, CST elevated the levels of UDPG and increased GYS2 activity. CST-KO mice had decreased liver glycogen that was restored by treatment with CST, reinforcing the crucial role of CST in hepatic glycogenesis. CST improved insulin signals downstream of IR and IRS-1 by enhancing phospho-AKT signals through the stimulation of PDK-1 and mTORC2 (mTOR Complex 2, rapamycin-insensitive complex) activities. CONCLUSIONS: CST directly promotes the glycogenic pathway by (a) reducing glucose production, (b) increasing glycogen synthesis from UDPG, (c) reducing glycogenolysis and (d) enhancing downstream insulin signalling.

Immunosuppression of Macrophages Underlies the Cardioprotective Effects of CST (Catestatin).

[Figure: see text].

Testicular germ cell tumors arise in the absence of sex-specific differentiation.

In response to signals from the embryonic testis, the germ cell intrinsic factor NANOS2 coordinates a transcriptional program necessary for the differentiation of pluripotent-like primordial germ cells toward a unipotent spermatogonial stem cell fate. Emerging evidence indicates that genetic risk factors contribute to testicular germ cell tumor initiation by disrupting sex-specific differentiation. Here, using the 129.MOLF-Chr19 mouse model of testicular teratomas and a NANOS2 reporter allele, we report that the developmental phenotypes required for tumorigenesis, including failure to enter mitotic arrest, retention of pluripotency and delayed sex-specific differentiation, were exclusive to a subpopulation of germ cells failing to express NANOS2. Single-cell RNA sequencing revealed that embryonic day 15.5 NANOS2-deficient germ cells and embryonal carcinoma cells developed a transcriptional profile enriched for MYC signaling, NODAL signaling and primed pluripotency. Moreover, lineage-tracing experiments demonstrated that embryonal carcinoma cells arose exclusively from germ cells failing to express NANOS2. Our results indicate that NANOS2 is the nexus through which several genetic risk factors influence tumor susceptibility. We propose that, in the absence of sex specification, signals native to the developing testis drive germ cell transformation.

Alternative RNA Splicing in Fatty Liver Disease.

Alternative RNA splicing is a process by which introns are removed and exons are assembled to construct different RNA transcript isoforms from a single pre-mRNA. Previous studies have demonstrated an association between dysregulation of RNA splicing and a number of clinical syndromes, but the generality to common disease has not been established. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease affecting one-third of adults worldwide, increasing the risk of cirrhosis and hepatocellular carcinoma (HCC). In this review we focus on the change in alternative RNA splicing in fatty liver disease and the role for splicing regulation in disease progression.

Time-restricted feeding normalizes hyperinsulinemia to inhibit breast cancer in obese postmenopausal mouse models.

Accumulating evidence indicates that obesity with its associated metabolic dysregulation, including hyperinsulinemia and aberrant circadian rhythms, increases the risk for a variety of cancers including postmenopausal breast cancer. Caloric restriction can ameliorate the harmful metabolic effects of obesity and inhibit cancer progression but is difficult to implement and maintain outside of the clinic. In this study, we aim to test a time-restricted feeding (TRF) approach on mouse models of obesity-driven postmenopausal breast cancer. We show that TRF abrogates the obesity-enhanced mammary tumor growth in two orthotopic models in the absence of calorie restriction or weight loss. TRF also reduces breast cancer metastasis to the lung. Furthermore, TRF delays tumor initiation in a transgenic model of mammary tumorigenesis prior to the onset of obesity. Notably, TRF increases whole-body insulin sensitivity, reduces hyperinsulinemia, restores diurnal gene expression rhythms in the tumor, and attenuates tumor growth and insulin signaling. Importantly, inhibition of insulin secretion with diazoxide mimics TRF whereas artificial elevation of insulin through insulin pumps implantation reverses the effect of TRF, suggesting that TRF acts through modulating hyperinsulinemia. Our data suggest that TRF is likely to be effective in breast cancer prevention and therapy.

Mitochondrial Dysfunction in Obesity and Reproduction.

Mounting evidence suggests a role for mitochondrial dysfunction in the pathogenesis of many diseases, including type 2 diabetes, aging, and ovarian failure. Because of the central role of mitochondria in energy production, heme biosynthesis, calcium buffering, steroidogenesis, and apoptosis signaling within cells, understanding the molecular mechanisms behind mitochondrial dysregulation and its potential implications in disease is critical. This review will take a journey through the past and summarize what is known about mitochondrial dysfunction in various disorders, focusing on metabolic alterations and reproductive abnormalities. Evidence is presented from studies in different human populations, and rodents with genetic manipulations of pathways known to affect mitochondrial function.

SIRT1 in Astrocytes Regulates Glucose Metabolism and Reproductive Function.

Sirtuin 1 (Sirt1) is an NAD-dependent class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, evidence suggests that SIRT1 in neurons plays a role in the central regulation of energy balance and reproduction, but no studies have addressed the contribution of astrocytes. We show here that overexpression of SIRT1 in astrocytes causes markedly increased food intake, body weight gain, and glucose intolerance, but expression of a deacetylase-deficient SIRT1 mutant decreases food intake and body weight and improves glucose tolerance, particularly in female mice. Paradoxically, the effect of these SIRT1 mutants on insulin tolerance was reversed, with overexpression showing greater insulin sensitivity. The mice overexpressing SIRT1 were more active, generated more heat, and had elevated oxygen consumption, possibly in compensation for the increased food intake. The female overexpressing mice were also more sensitive to diet-induced obesity. Reproductively, the mice expressing the deacetylase-deficient SIRT1 mutant had impaired estrous cycles, decreased LH surges, and fewer corpora lutea, indicating decreased ovulation. The GnRH neurons were responsive to kisspeptin stimulation, but hypothalamic expression of Kiss1 was reduced in the mutant mice. Our results showed that SIRT1 signaling in astrocytes can contribute to metabolic and reproductive regulation independent of SIRT1 effects in neurons.

Neuronal SIRT1 Regulates Metabolic and Reproductive Function and the Response to Caloric Restriction.

Sirt1 is an NAD-dependent, class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. In this study, we generated mice expressing an enzymatically inactive form (N-MUT) or wild-type (WT) SIRT1 (N-OX) in mature neurons. N-OX male and female mice had impaired glucose tolerance, and N-MUT female, but not male, mice had improved glucose tolerance compared with that of WT littermates. Furthermore, glucose tolerance was improved in all mice with caloric restriction (CR) but was greater in the N-OX mice, who had better glucose tolerance than their littermates. At the reproductive level, N-OX females had impaired estrous cycles, with increased cycle length and more time in estrus. LH and progesterone surges were absent on the evening of proestrus in the N-OX mice, suggesting a defect in spontaneous ovulation, which was confirmed by the ovarian histology revealing fewer corpora lutea. Despite this defect, the mice were still fertile when mated to WT mice on the day of proestrus, indicating that the mice could respond to normal pheromonal or environmental cues. When subjected to CR, the N-OX mice went into diestrus arrest earlier than their littermates. Together, these results suggested that the overexpression of SIRT1 rendered the mice more sensitive to the metabolic improvements and suppression of reproductive cycles by CR, which was independent of circadian rhythms.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 µM) and PACAP (0.1 µM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 µM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.