Endothelial Nitric Oxide Synthase-Deficient Mice: A Model of Spontaneous Cerebral Small-Vessel Disease.

Age-related cerebral small-vessel disease (CSVD) is a major cause of stroke and dementia. Despite a widespread acceptance of small-vessel arteriopathy, lacunar infarction, diffuse white matter injury, and cognitive impairment as four cardinal features of CSVD, a unifying pathologic mechanism of CSVD remains elusive. Herein, we introduce partial endothelial nitric oxide synthase (eNOS)-deficient mice as a model of age-dependent, spontaneous CSVD. These mice developed cerebral hypoperfusion and blood-brain barrier leakage at a young age, which progressively worsened with advanced age. Their brains exhibited elevated oxidative stress, astrogliosis, cerebral amyloid angiopathy, microbleeds, microinfarction, and white matter pathology. Partial eNOS-deficient mice developed gait disturbances at middle age, and hippocampus-dependent memory deficits at older ages. These mice also showed enhanced expression of bone morphogenetic protein 4 (BMP4) in brain pericytes before myelin loss and white matter pathology. Because BMP4 signaling not only promotes astrogliogenesis but also blocks oligodendrocyte differentiation, we posit that paracrine actions of BMP4, localized within the neurovascular unit, promote white matter disorganization and neurodegeneration. These observations point to BMP4 signaling pathway in the aging brain vasculature as a potential therapeutic target. Finally, because studies in partial eNOS-deficient mice corroborated recent clinical evidence that blood-brain barrier disruption is a primary cause of white matter pathology, the mechanism of impaired nitric oxide signaling-mediated CSVD warrants further investigation.

Insulin-induced de novo lipid synthesis occurs mainly via mTOR-dependent regulation of proteostasis of SREBP-1c.

Insulin stimulates de novo lipid synthesis in the liver and in cultured hepatocytes via its ability to activate sterol regulatory element-binding protein 1c (SREBP-1c). Although PI3K-AKT-mTORC1-p70S6K-signaling kinases are known to drive feed-forward expression of SREBP-1c, the identity of the phosphorylated amino acid residue(s) putatively involved in insulin-stimulated de novo lipogenesis remains elusive. We obtained in silico and mass spectrometry evidence, that was combined with siRNA strategies, to discover that insulin-induced phosphorylation of serine 418, serine 419, and serine 422 in rat SREBP-1c was most likely mediated by p70S6 kinase. Here, for the first time, we show that insulin-induced phosphorylation of these 3 serine residues mainly impinged on the mechanisms of proteostasis of both full-length and mature SREBP-1c in the McArdle-RH7777 hepatoma cells. Consistent with this conclusion, nascent SREBP-1c, substituted with phosphomimetic aspartic acid residues at these 3 sites, was resistant to proteasomal degradation. As a consequence, endoplasmic reticulum to Golgi migration and proteolytic maturation of pSREBP-1c was significantly enhanced which led to increased accumulation of mature nSREBP-1c, even in the absence of insulin. Remarkably, aspartic acid substitutions at S418, S419 and S422 also protected the nascent SREBP-1c from ubiquitin-mediated proteasome degradation thus increasing its steady-state levels and transactivation potential in the nucleus. These complementary effects of p70S6K-mediated phosphorylation on proteostasis of pSREBP-1c were necessary and sufficient to account for insulin's ability to enhance transcription of genes controlling de novo lipogenesis in hepatocytes.

Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.

Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI. Using miRCURY LNA™ Universal microRNA PCR system, we demonstrate that H9c2 cells exposed to TSA for 6 and 24 h elicited differential expression of 19 and 16 microRNAs, respectively. H9c2 cells incubated in medium-containing 100 nM of TSA elicited a rapid and robust induction of miR-129-5p. Enhanced expression of miR-129-5p was also observed in the hearts of TSA-treated mice. Induction of miR-129-5p in H9c2 cells was accompanied by reduced expression of its direct target, cyclin-dependent kinase 6 (CDK6) that is a key regulator of cell cycle. Using cell division-dependent dilution of Cell Trace™ violet measurements we showed that concomitant induction of miR-129-5p and reduced CDK6 expression were mechanistically involved in TSA-induced inhibition of proliferation of H9c2 cells. Consistent with this scenario, cells expressing an antagomiR of miR-129-5p were resistant to the anti-proliferative actions of TSA. These data indicate that although TSA treatment led to altered expression of several microRNAs, the overarching action of TSA (i.e., inhibition of cell division) in H9c2 cells was achieved via miR-129-5p.

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

Phosphorylation of sterol regulatory element binding protein-1a by protein kinase A (PKA) regulates transcriptional activity.

The counter-regulatory hormone glucagon inhibits lipogenesis via downregulation of sterol regulatory element binding protein 1 (SREBP-1). The effect of glucagon is mediated via protein kinase A (PKA). To determine if SREBP-1 is a direct phosphorylation target of PKA, we conducted mass spectrometry analysis of recombinant n-terminal SREBP-1a following PKA treatment in vitro. This analysis identified serines 331/332 as bona-fide phosphorylation targets of PKA. To determine the functional consequences of phosphorylation at these sites, we constructed mammalian expression vector for both nSREBP-1a and 1c isoforms in which the candidate PKA phosphorylation sites were mutated to active phosphomimetic or non-phosphorylatable amino acids. The transcriptional activity of SREBP was reduced by the phosphomimetic mutation of S332 of nSREBP-1a and the corresponding serine (S308) of nSREBP-1c. This site is a strong candidate for mediating the negative regulatory effect of glucagon on SREBP-1 and lipogenesis.

FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c.

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ∼60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ∼2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via "feed forward" regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

Effect of statin treatment on metabolites, lipids and prostanoids in patients with Statin Associated Muscle Symptoms (SAMS).

BACKGROUND: Between 5-10% of patients discontinue statin therapy due to statin-associated adverse reactions, primarily statin associated muscle symptoms (SAMS). The absence of a clear clinical phenotype or of biomarkers poses a challenge for diagnosis and management of SAMS. Similarly, our incomplete understanding of the pathogenesis of SAMS hinders the identification of treatments for SAMS. Metabolomics, the profiling of metabolites in biofluids, cells and tissues is an important tool for biomarker discovery and provides important insight into the origins of symptomatology. In order to better understand the pathophysiology of this common disorder and to identify biomarkers, we undertook comprehensive metabolomic and lipidomic profiling of plasma samples from patients with SAMS who were undergoing statin rechallenge as part of their clinical care. METHODS AND FINDINGS: We report our findings in 67 patients, 28 with SAMS (cases) and 39 statin-tolerant controls. SAMS patients were studied during statin rechallenge and statin tolerant controls were studied while on statin. Plasma samples were analyzed using untargeted LC-MS metabolomics and lipidomics to detect differences between cases and controls. Differences in lipid species in plasma were observed between cases and controls. These included higher levels of linoleic acid containing phospholipids and lower ether lipids and sphingolipids. Reduced levels of acylcarnitines and altered amino acid profile (tryptophan, tyrosine, proline, arginine, and taurine) were observed in cases relative to controls. Pathway analysis identified significant increase of urea cycle metabolites and arginine and proline metabolites among cases along with downregulation of pathways mediating oxidation of branched chain fatty acids, carnitine synthesis, and transfer of acetyl groups into mitochondria. CONCLUSIONS: The plasma metabolome of patients with SAMS exhibited reduced content of long chain fatty acids and increased levels of linoleic acid (18:2) in phospholipids, altered energy production pathways (ß-oxidation, citric acid cycle and urea cycles) as well as reduced levels of carnitine, an essential mediator of mitochondrial energy production. Our findings support the hypothesis that alterations in pro-inflammatory lipids (arachidonic acid pathway) and impaired mitochondrial energy metabolism underlie the muscle symptoms of patients with statin associated muscle symptoms (SAMS).

Pan-histone deacetylase inhibitors regulate signaling pathways involved in proliferative and pro-inflammatory mechanisms in H9c2 cells.

BACKGROUND: We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA. RESULTS: We incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGFß, TNF-α, IFNγ and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGFß- and TNF-α-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-α and IFNγ specific networks at 6 h, followed by elicitation of IL-6 and IFNγ-centered gene networks at 24h. CONCLUSIONS: Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically.

Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy.

We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles. Of 184 genes that were differentially regulated by IL-18 and TSA, 33 were regulated in an opposite manner. The hearts of mice treated with IL-18 and/or CBHA elicited 147 differentially expressed genes (DEGs), a third of which were oppositely regulated by IL-18 and CBHA. Ingenuity Pathways and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs showed that IL-18 impinged on TNF-α- and IFNγ-specific gene networks relegated to controlling immunity and inflammation, cardiac metabolism and energetics, and cell proliferation and apoptosis. These TNF-α- and IFNγ-specific gene networks, extensively connected with PI3K, MAPK, and NF-κB signaling pathways, were oppositely regulated by IL-18 and pan-HDACIs. Evidently, both TSA and CBHA caused a two- to fourfold induction of phosphatase and tensin homolog expression to counteract IL-18-induced proinflammatory signaling and cardiac hypertrophy.

Insulin enhances the biogenesis of nuclear sterol regulatory element-binding protein (SREBP)-1c by posttranscriptional down-regulation of Insig-2A and its dissociation from SREBP cleavage-activating protein (SCAP).SREBP-1c complex.

The regulation of lipid homeostasis by insulin is mediated in part by the enhanced transcription of the gene encoding sterol regulatory element-binding protein-1c (SREBP-1c). The nascent SREBP-1c is embedded in the endoplasmic reticulum (ER) and must be transported to the Golgi where two sequential cleavages generate its NH(2)-terminal fragment, nSREBP-1c. We have shown recently that in primary cultures of rat hepatocytes, insulin rapidly and selectively stimulates proteolytic processing of the nascent SREBP-1c by enhancing the affinity of the SREBP cleavage-activating protein (SCAP).SREBP-1c complex for coatomer protein complex II (COPII) vesicles. The SCAP.SREBP complex is retained in the ER by Insig proteins. We report here that insulin persistently stimulates controlled proteolysis of the nascent SREBP-1c by selectively reducing the level of Insig-2a protein via accelerated degradation of its cognate mRNA. Insulin enhanced the rate of turnover of Insig-2a mRNA via its 3'-untranslated region. Insulin-induced depletion of Insig-2a promotes association of the SCAP.SREBP-1c complex with COPII vesicles and subsequent migration to the Golgi where site-1 and site-2 proteases process the nascent SREBP-1c. Consistent with this mechanism, experimental knockdown of Insig-2a expression with small interfering RNA mimicked insulin-induced proteolysis of the nascent SREBP-1c, whereas exogenous expression of Insig-2a in hepatocytes led to reduced intramembrane proteolysis of the newly synthesized SREBP-1c. The action of insulin on the processing of the nascent SREBP-1c via Insig-2a was highly selective, as proteolysis of the newly synthesized SREBP-2 remained unchanged under identical conditions. On the basis of these data, we propose that the stimulation of SREBP-1c processing by insulin is mediated by a selective depletion of Insig-2a protein by promoting decay of its cognate mRNA. Thus, insulin-induced reduction in Insig-2a protein leads to an enhanced export of the SCAP.SREBP-1c complex from ER to the Golgi.

N-3 polyunsaturated fatty acids suppress insulin-induced SREBP-1c transcription via reduced trans-activating capacity of LXRalpha.

Insulin coordinately up-regulates lipogenic gene transcription via induction of sterol regulatory element binding protein-1c (SREBP-1c). Conversely, polyunsaturated fatty acids (PUFA) decrease lipogenic gene transcription via suppression of SREBP-1c. We therefore examined the ability of n-3 PUFA to mitigate induction of SREBP-1c and its downstream lipogenic targets by insulin in primary rat hepatocyte cultures. Insulin induced expression of SREBP-1c mRNA 5-6 fold as well as rat SREBP-1c promoter activity. These effects were prevented by the n-3 fatty acids eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3, DHA), but not by the monounsaturated fatty acid oleic acid (18:1 n-6, OLA). N-3 fatty acids also effectively prevented insulin induction of the downstream lipogenic enzyme targets fatty acid synthase (FAS) and acetyl carboxyl coenzyme acetyltransferase-1 (ACC-1), and reduced de novo lipogenesis. The SREBP-1c promoter contains an insulin response unit consisting of tandem LXRalpha response elements (LXREs) as well as sites for NF-Y, Sp1, and SREBP-1c itself. The LXREs were identified as a primary site mediating suppression of SREBP-1c transcription by n-3 PUFA. DHA effectively prevented LXRalpha-dependent activation of both the wild type SREBP-1c promoter and the synthetic LXRE-driven promoter, and significantly blunted LXRalpha-dependent activation of a Gal4-LXRalpha chimeric protein thus demonstrating that n-3 PUFA effectively mitigate induction of SREBP-1c by insulin via reduced trans-activation of LXRalpha.

SREBPs: the crossroads of physiological and pathological lipid homeostasis.

The uptake, biosynthesis and metabolism of cholesterol and other lipids are exquisitely regulated by feedback and feed-forward pathways in organisms ranging from Caenorhabditis elegans to humans. As endoplasmic reticulum (ER) membrane-embedded transcription factors that are activated in the Golgi apparatus, sterol regulatory element-binding proteins (SREBPs) are central to the intracellular surveillance of lipid catabolism and de novo biogenesis. The biosynthesis of SREBP proteins, their migration from the ER to the Golgi compartment, intra-membrane proteolysis, nuclear translocation and trans-activation potential are tightly controlled in vivo. Here we summarize recent studies elucidating the transcriptional and post-transcriptional regulation of SREBP-1c through nutrition and the action of hormones, particularly insulin, and the resulting implications for dyslipidemia of obesity, metabolic syndrome and type 2 diabetes.

Phosphorylation dependent proteostasis of sterol regulatory element binding proteins.

Lipid homeostasis is critically dependent on the liver. Hepatic genes involved in lipid biosynthesis are controlled by combinatorial actions of multiple transcription factors that include three sterol regulatory element binding proteins (SREBPs), carbohydrate responsive element binding protein, liver X receptors, and others. SREBP-1c, a seminal regulator of de novo lipogenesis, resides in the endoplasmic reticulum as a transcriptionally inert precursor and must undergo a regulated intramembrane proteolysis (RIP) prior to its nuclear translocation as a bone fide transcription factor. The regulation of biosynthesis, turnover and actions of SREBP-1c and lipogenesis are mechanistically linked to signaling kinases, canonically induced by macronutrients and insulin. Here, we briefly review the evidence showing that phosphorylation of SREBP-1c and its interacting partners, catalyzed by phosphatidyl inositol-3-kinase, protein kinase B, mechanistic target of rapamycin complex 1 and 2, mitogen activated protein kinases, glycogen synthase kinase-3ß, protein kinase A and 5' adenosine monophosphate-activated protein kinase regulates the mechanisms of RIP and stability of SREBP-1c and de novo lipogenesis.

A critical role of Sp1 transcription factor in regulating gene expression in response to insulin and other hormones.

Specificity protein 1 (Sp1) belongs to a family of ubiquitously expressed, C(2)H(2)-type zinc finger-containing DNA binding proteins that activate or repress transcription of many genes in response to physiological and pathological stimuli. There is emerging evidence to indicate that in addition to functioning as 'housekeeping' transcription factors, members of Sp family may be key mediators of gene expression induced by insulin and other hormones. The founding member of the family, Sp1, by virtue of its multi-domain organization, potential for posttranslational modifications and interactions with numerous transcription factors, represents an ideal mediator of nuclear signaling in response to hormones. Insulin regulates the sub-cellular localization, stability and trans-activation potential of Sp1 by dynamically modulating its post-translational modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) or phosphate residues. We briefly review the recent literature demonstrating that an involvement of Sp-family of transcription factors in the regulation of differential gene expression in response to hormones is more common than previously appreciated and may represent a key regulatory mechanism.

Circadian rhythms of hormone secretion and obesity.

The adipose tissue homeostasis is profoundly affected by circadian rhythms of corticosteroid secretion and chronic loss of hormonal oscillations is associated with obesity. How adipose tissue differentially responds to pulsatile vs continuous presence of glucocorticoids is poorly defined. To address this question, Bahrami-Nejad et al studied differentiation of pre-adipocytes, containing endogenously tagged CCAAT/enhancer binding protein and peroxisome proliferator-activated receptor (PPAR) γ (key regulators of adipocyte differentiation), in response to corticosteroids that were delivered either in an oscillatory fashion or continuously. The authors show that the bi-stable state of differentiation of pre-adipocytes and adipocytes was regulated by a combination of fast and slow positive feedback networks, that determined unique threshold of PPARγ in these cells. Evidently, pre-adipocytes used the fast feedback loop to reject differentiation cues of oscillating pulses of glucocorticoids and failed to differentiate into fat cells. In contrast, when glucocorticoids were delivered continuously, precursor cells exploited the slow feedback loop to embark on a path of maximal differentiation. This differential differentiation response of pre-adipocytes to pulsatile vs continuous exposure to glucocorticoids was corroborated in vivo. Thus, mice receiving non-oscillating doses of exogenous glucocorticoids, for 21 d, elicited excessive accumulation of visceral and subcutaneous fat. These data shed new light on the mechanisms of obesity caused by putative misalignment of circadian secretion of glucocorticoids or their persistently high levels due to chronic stress or Cushing's disease.

Sex-specific differences in hepatic steatosis in obese spontaneously hypertensive (SHROB) rats.

BACKGROUND: Patients with metabolic syndrome, who are characterized by co-existence of insulin resistance, hypertension, hyperlipidemia, and obesity, are also prone to develop non-alcoholic fatty liver disease (NAFLD). Although the prevalence and severity of NAFLD is significantly greater in men than women, the mechanisms by which gender modulates the pathogenesis of hepatic steatosis are poorly defined. The obese spontaneously hypertensive (SHROB) rats represent an attractive model of metabolic syndrome without overt type 2 diabetes. Although pathological manifestation caused by the absence of a functional leptin receptor has been extensively studied in SHROB rats, it is unknown whether these animals elicited sex-specific differences in the development of hepatic steatosis. METHODS: We compared hepatic pathology in male and female SHROB rats. Additionally, we examined key biochemical and molecular parameters of signaling pathways linked with hyperinsulinemia and hyperlipidemia. Finally, using methods of quantitative polymerase chain reaction (qPCR) and western blot analysis, we quantified expression of 45 genes related to lipid biosynthesis and metabolism in the livers of male and female SHROB rats. RESULTS: We show that all SHROB rats developed hepatic steatosis that was accompanied by enhanced expression of SREBP1, SREBP2, ACC1, and FASN proteins. The livers of male rats also elicited higher induction of Pparg, Ppara, Slc2a4, Atox1, Skp1, Angptl3, and Pnpla3 mRNAs. In contrast, the livers of female SHROB rats elicited constitutively higher levels of phosphorylated JNK and AMPK and enhanced expression of Cd36. CONCLUSION: Based on these data, we conclude that the severity of hepatic steatosis in male and female SHROB rats was mainly driven by increased de novo lipogenesis. Moreover, male and female SHROB rats also elicited differential severity of hepatic steatosis that was coupled with sex-specific differences in fatty acid transport and esterification.

Gut-brain crosstalk regulates craving for fatty food.

Patients undergoing Roux-en-Y gastric bypass (RYGB) surgery elicit striking loss of body weight. Anatomical re-structuring of the gastrointestinal (GI) tract, leading to reduced caloric intake and changes in food preference, are thought to be the primary drivers of weight loss in bariatric surgery patients. However, the mechanisms by which RYGB surgery causes a reduced preference for fatty foods remain elusive. In a recent report, Hankir et al described how RYGB surgery modulated lipid nutrient signals in the intestine of rats to blunt their craving for fatty food. The authors reported that RYGB surgery restored an endogenous fat-satiety signaling pathway, mediated via oleoylethanolamide (OEA), that was greatly blunted in obese animals. In RYGB rats, high fat diet (HFD) led to increased production of OEA that activated the intestinal peroxisome proliferation activator receptors-α (PPARα). In RYGB rats, activation of PPARα by OEA was accompanied by enhanced dopamine neurotransmission in the dorsal striatum and reduced preference for HFD. The authors showed that OEA-mediated signals to the midbrain were transmitted via the vagus nerve. Interfering with either the production of OEA in enterocytes, or blocking of vagal and striatal D1 receptors signals eliminated the decreased craving for fat in RYGB rats. These studies demonstrated that bariatric surgery led to alterations in the reward circuitry of the brain in RYGB rats and reduced their preference for HFD.

Statins redux: A re-assessment of how statins lower plasma cholesterol.

Obesity associated dyslipidemia and its negative effects on the heart and blood vessels have emerged as a major healthcare challenge around the globe. The use of statins, potent inhibitors of hydroxyl-methyl glutaryl (HMG) Co-A reductase, a rate-limiting enzyme in cholesterol biosynthesis, has significantly reduced the rates of cardiovascular and general mortality in patients with coronary artery disease. How statins lower plasma cholesterol levels presents a mechanistic conundrum since persistent exposure to these drugs in vitro or in vivo is known to induce overexpression of the HMG Co-A reductase gene and protein. In an attempt to solve this mechanistic puzzle, Schonewille et al, studied detailed metabolic parameters of cholesterol synthesis, inter-organ flux and excretion in mice treated with 3 common statins, rosuvastatin, atorvastatin or lovastatin, each with its unique pharmacokinetics. From the measurements of the rates of heavy water (D2O) and [13C]-acetate incorporation into lipids, the authors calculated the rates of whole body and organ-specific cholesterol synthesis in control and statin-treated mice. These analyses revealed dramatic enhancement in the rates of hepatic cholesterol biosynthesis in statin-treated mice that concomitantly elicited lower levels of cholesterol in their plasma. The authors have provided strong evidence to indicate that statin treatment in mice led to induction of compensatory metabolic pathways that apparently mitigated an excessive accumulation of cholesterol in the body. It was noted however that changes in cholesterol metabolism induced by 3 statins were not identical. While sustained delivery of all 3 statins led to enhanced rates of biliary excretion of cholesterol and its fecal elimination, only atorvastatin treated mice elicited enhanced trans-intestinal cholesterol excretion. Thus, blockade of HMGCR by statins in mice was associated with profound metabolic adaptations that reset their cholesterol homeostasis. The findings of Schonewille et al, deserve to be corroborated and extended in patients in order to more effectively utilize these important cholesterol-lowering drugs in the clinic.

Patients experiencing statin-induced myalgia exhibit a unique program of skeletal muscle gene expression following statin re-challenge.

Statins, the 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitors, are widely prescribed for treatment of hypercholesterolemia. Although statins are generally well tolerated, up to ten percent of statin-treated patients experience myalgia symptoms, defined as muscle pain without elevated creatinine phosphokinase (CPK) levels. Myalgia is the most frequent reason for discontinuation of statin therapy. The mechanisms underlying statin myalgia are not clearly understood. To elucidate changes in gene expression associated with statin myalgia, we compared profiles of gene expression in skeletal muscle biopsies from patients with statin myalgia who were undergoing statin re-challenge (cases) versus those of statin-tolerant controls. A robust separation of case and control cohorts was revealed by Principal Component Analysis of differentially expressed genes (DEGs). To identify putative gene expression and metabolic pathways that may be perturbed in skeletal muscles of patients with statin myalgia, we subjected DEGs to Ingenuity Pathways (IPA) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analyses. The most prominent pathways altered by statins included cellular stress, apoptosis, cell senescence and DNA repair (TP53, BARD1, Mre11 and RAD51); activation of pro-inflammatory immune response (CXCL12, CST5, POU2F1); protein catabolism, cholesterol biosynthesis, protein prenylation and RAS-GTPase activation (FDFT1, LSS, TP53, UBD, ATF2, H-ras). Based on these data we tentatively conclude that persistent myalgia in response to statins may emanate from cellular stress underpinned by mechanisms of post-inflammatory repair and regeneration. We also posit that this subset of individuals is genetically predisposed to eliciting altered statin metabolism and/or increased end-organ susceptibility that lead to a range of statin-induced myopathies. This mechanistic scenario is further bolstered by the discovery that a number of single nucleotide polymorphisms (e.g., SLCO1B1, SLCO2B1 and RYR2) associated with statin myalgia and myositis were observed with increased frequency among patients with statin myalgia.