Circadian clock gene disruption in white blood cells of patients with celiac disease.

Clock gene disruption has been reported in inflammatory and autoimmune diseases. Specifically, it has been shown that clock gene expression is down-regulated in intestinal tissue and peripheral blood mononuclear cells of patients with inflammatory bowel disease (IBD). We aimed to determine the systemic expression of the circadian clock genes in newly diagnosed untreated, young patients with celiac disease (CeD). We prospectively enrolled patients younger than 20 years old who underwent diagnostic endoscopic procedures either for CeD diagnosis or due to other gastrointestinal complaints, at the pediatric and adult gastroenterology units, the Tel Aviv Sourasky Medical Center from 8/2016-8/2022. Demographic data, anthropometric parameters, and endoscopic macroscopic and microscopic findings were obtained. Blood samples were obtained to determine tissue transglutaminase (tTG) and core clock gene (CLOCK, BMAL1, PER1, PER2, CRY1, CRY2) expression in white blood cells (WBC). Thirty individuals were analyzed (18 with newly diagnosed CeD and 12 controls). Expression of the clock genes CLOCK, BMAL1, CRY2, PER1 and PER2 was significantly reduced in CeD patients compared to controls, while CRY1 did not differ between the groups. In conclusion, newly diagnosed, untreated, young patients with CeD have reduced clock gene expression in WBC compared to controls. These results suggest that, in CeD, the inflammatory response is associated with systemic disruption of clock gene expression, as is manifested in other inflammatory and autoimmune diseases. CLINICALTRIALS.GOV IDENTIFIER: NCT03662646.

Inverse Relationship Between Clock Gene Expression and Inflammatory Markers in Ulcerative Colitis Patients Undergoing Remission.

BACKGROUND: Changes in the expression of clock genes have been reported in inflammatory bowel disease (IBD) patients. AIMS: We aimed to investigate whether reduced inflammation restores clock gene expression to levels of healthy controls. METHODS: This was a prospective study. Participants completed questionnaires providing data on demographics, sleeping habits, and disease activity. Anthropometric parameters, C-reactive protein (CRP), and fecal calprotectin (Fcal) levels were collected. Peripheral blood samples were analyzed for clock gene (CLOCK, BMAL1, CRY1, CRY2, PER1, PER2) expression. Patients with IBD were separated by diagnosis into ulcerative colitis (UC) and Crohn's disease (CD). Each diagnosis was further divided into active disease and disease under remission. RESULTS: Forty-nine patients with IBD and 19 healthy controls completed the study. BMAL1 and PER2 were significantly reduced in active patients with UC compared to patients with UC in remission. BMAL1, PER1, and PER2 were significantly reduced in patients with UC with CRP > 5 mg/dl. PER2, CRY1, and CRY2 were significantly reduced in patients with UC with Fcal > 250 mg/kg. Clock gene expression of patients with UC in remission was comparable to healthy controls. When all patients with IBD were analyzed, an overshoot in CRY1 expression was observed in patients in remission, patients with CRP < 5 mg/dl, and patients with Fcal < 250 mg/kg. CONCLUSION: CRP and Fcal are inversely related to clock gene levels in patients with UC. CRY1 may play a role in counteracting the anti-inflammatory processes when remission is induced in patients with IBD. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03662646.

Circadian rhythms, nutrition and implications for longevity in urban environments.

Presently, about 12% of the population is 65 years or older and by the year 2030 that figure is expected to reach 21%. In order to promote the well-being of the elderly and to reduce the costs associated with health care demands, increased longevity should be accompanied by ageing attenuation. Energy restriction, which limits the amount of energy consumed to 60-70% of the daily intake, and intermittent fasting, which allows the food to be available ad libitum every other day, extend the life span of mammals and prevent or delay the onset of major age-related diseases, such as cancer, diabetes and cataracts. Recently, we have shown that well-being can be achieved by resetting of the circadian clock and induction of robust catabolic circadian rhythms via timed feeding. In addition, the clock mechanism regulates metabolism and major metabolic proteins are key factors in the core clock mechanism. Therefore, it is necessary to increase our understanding of circadian regulation over metabolism and longevity and to design new therapies based on this regulation. This review will explore the present data in the field of circadian rhythms, ageing and metabolism.

Serotonin prevents differentiation into brown adipocytes and induces transdifferentiation into white adipocytes.

BACKGROUND/OBJECTIVES: Serotonin is synthesized by many cells in the periphery to affect vasoconstriction, intestinal motility, and glucose and lipid metabolism. It has recently been shown that serotonin leads to fat accumulation in white adipose tissue (WAT). However, the direct effect of serotonin on brown adipose tissue differentiation and metabolism is limited. Therefore, our aim was to investigate the effect of serotonin on brown adipocyte metabolism and differentiation. METHODS: Non-differentiated HIB1B cells and differentiated HIB1B brown adipocytes were treated with serotonin and their metabolism and differentiation examined. RESULTS: Differentiated HIB1B brown adipocytes treated with serotonin had reduced levels of the thermogenic markers uncoupling protein 1 (UCP1) and fibroblast growth factor 21 (FGF21) and increased levels of UCP2. In parallel, serotonin led to 3-6-fold reduction in the gene expression of brown adipocyte differentiation markers, that is, Prdm16 (positive regulatory domain 16), Bmp7 (bone morphogenic protein 7) and Pparγ (peroxisome-proliferator-activated receptor γ). Serotonin treatment reduced catabolism and mitochondrial activity shifting metabolism towards fatty acid synthesis rather than oxidation. Strikingly, non-differentiated HIB1B preadipocytes incubated with serotonin failed to differentiate into brown adipocytes. Moreover, although BMP6-treated myoblasts can readily differentiate into brown adipocytes, serotonin interfered with this process. CONCLUSIONS: Serotonin leads to whitening of brown adipocytes, shifting their metabolism to fat accumulation rather than oxidation. In addition, serotonin interferes with the differentiation process into brown adipocytes.

Peroxisome proliferator-activated receptor α (PPARα) activation advances locomotor activity and feeding daily rhythms in mice.

Peroxisome proliferator-activated receptors (PPARs) are key mediators of energy homeostasis, and lipid and glucose metabolism that exhibit circadian expression. PPAR activating drugs are used clinically as lipid and glucose-lowering drugs. We evaluated the effect of long-term (11 weeks) PPARα and PPARγ activation using bezafibrate and rosiglitazone, respectively, on metabolism, locomotor activity and feeding rhythms of non-obese mice. We found that bezafibrate, but not rosiglitazone, led to no weight gain and a slight weight loss with reduced epididymal fat pads. Although rosiglitazone had a minor effect on 24-h food intake rhythm, bezafibrate treatment was accompanied by increased amplitude and an advanced acrophase of the 24-h feeding rhythm. Similarly, unlike rosiglitazone, bezafibrate treatment was accompanied by a significantly advanced acrophase of locomotor activity rhythm under constant darkness conditions. As disrupted circadian rhythms lead to obesity, PPARα activation can serve as a clinical target for the modulation of both circadian rhythms and metabolism.

Spontaneous caloric restriction associated with increased leptin levels in obesity-resistant αMUPA mice.

BACKGROUND: αMUPA mice carry as a transgene the cDNA encoding urokinase-type plasminogen activator, a member of the plasminogen/plasmin system that functions in fibrinolysis and extracellular proteolysis. These mice spontaneously consume less food when fed ad libitum and live longer compared with wild-type (WT) control mice. αMUPA mice are obesity resistant and they share many similarities with calorically restricted animals. However, extensive metabolic characterization of this unique transgenic model has never been performed. METHOD: Metabolism of αMUPA mice was analyzed by measuring hormone, lipid and glucose levels in the serum, as well as gene and protein expression levels in the liver, hypothalamus and brainstem. RESULTS: αMUPA mice were found to be leaner than WT mice mainly because of reduced fat depots. Serum analyses showed that αMUPA mice have high levels of the anorexigenic hormones insulin and leptin, and low levels of the orexigenic hormone ghrelin. Analyses of brain neuropeptides showed that the transcript of the anorexigenic neuropeptide Pomc is highly expressed in the brainstem, whereas the expression of the orexigenic neuropeptides Npy, Orexin and Mch is blunted in the hypothalamus of αMUPA mice. In addition, adenosine monophosphate (AMP)-activated protein kinase (AMPK) levels were higher in the liver and lower in the hypothalamus, thus promoting simultaneously central reduction in appetite and peripheral loss of fat. The levels of SIRT1 were low in the liver, but high in the hypothalamus, a feature that αMUPA mice share with calorically restricted animals. CONCLUSION: Taken together, αMUPA mice exhibit a unique metabolic phenotype of low-calorie intake and high leptin levels, and could serve as a model for both spontaneous calorie restriction and resistance to obesity.

The suprachiasmatic nuclei are involved in determining circadian rhythms during restricted feeding.

The circadian clock in the suprachiasmatic nuclei (SCN) responds to light and regulates peripheral circadian rhythms. Feeding regimens also reset the clock, so that time-restricted feeding (RF) dictates rhythms in peripheral tissues, whereas calorie restriction (CR) affects the SCN clock. To better understand the influence of RF vs. CR on circadian rhythms, we took advantage of the transgenic alphaMUPA mice that exhibit spontaneously reduced eating, and can serve as a model for CR under ad libitum feeding, and a model for temporal CR under RF compared with wild type (WT) mice. Our results show that RF advanced and generally increased the amplitude of clock gene expression in the liver under LD in both mouse types. However, under disruptive light conditions, RF resulted in a different clock gene phase in WT mice compared with alphaMUPA mice, suggesting a role for the reduced calories in resetting the SCN that led to the change of phase in alphaMUPA mice. Comparison of the RF regimen in the two lighting conditions in WT mice revealed that mPer1, mClock, and mBmal1 increased, whereas mPer2 decreased in amplitude under ultradian light in WT mice, suggesting a role for the SCN in determining clock gene expression in the periphery during RF. In summary, herein we reinforce a role for calorie restriction in resetting the SCN clock, and unravel a role for the SCN in determining peripheral rhythms under RF.

Diversification of neurotoxins by C-tail 'wiggling': a scorpion recipe for survival.

The structure of bioactive surfaces of proteins is a subject of intensive research, yet the mechanisms by which such surfaces have evolved are largely unknown. Polypeptide toxins produced by venomous animals such as sea anemones, cone snails, scorpions, and snakes show multiple routes for active site diversification, each maintaining a typical conserved scaffold. Comparative analysis of an array of genetically related scorpion polypeptide toxins that modulate sodium channels in neuronal membranes suggests a unique route of toxic site diversification. This premise is based on recent identification of bioactive surfaces of toxin representative of three distinct pharmacological groups and a comparison of their 3-dimensional structures. Despite their similar scaffold, the bioactive surfaces of the various toxins vary considerably, but always coincide with the molecular exterior onto which the C-tail is anchored. Superposition of the toxin structures indicates that the C-tails diverge from a common structural start point, which suggests that the pharmacological versatility displayed by these toxins might have been achieved along evolution via structural reconfiguration of the C-tail, leading to reshaping of new bioactive surfaces.

Structural implications on the interaction of scorpion alpha-like toxins with the sodium channel receptor site inferred from toxin iodination and pH-dependent binding.

The alpha-like toxin from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh III) binds with high affinity to receptor site 3 on insect sodium channels but does not bind to rat brain synaptosomes. The binding affinity of Lqh III to cockroach neuronal membranes was fivefold higher at pH 6.5 than at pH 7.5. This correlated with an increase in the electropositive charge on the toxin surface resulting from protonation of its four histidines. Radioiodination of Tyr(14) of Lqh III abolished its binding to locust but not cockroach sodium channels, whereas the noniodinated toxin bound equally well to both neuronal preparations. Radioiodination of Tyr(10) or Tyr(21) of the structurally similar alpha-toxin from L. quinquestriatus hebraeus (LqhalphaIT), as well as their substitution by phenylalanine, had only minor effects on binding to cockroach neuronal membranes. However, substitution of Tyr(21), but not Tyr(14), by leucine decreased the binding affinity of LqhalphaIT approximately 87-fold. Thus, Tyr(14) is involved in the bioactivity of Lqh III to locust receptor site 3 and is not crucial for the binding of LqhalphaIT to this site. In turn, the aromatic ring of Tyr(21) takes part in the bioactivity of LqhalphaIT to insects. These results highlight subtle architectural variations between locust and cockroach receptor site 3, in addition to previous results demonstrating the competence of Lqh III to differentiate between insect and mammalian sodium channel subtypes.

The putative bioactive surface of insect-selective scorpion excitatory neurotoxins.

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

Dynamic diversification from a putative common ancestor of scorpion toxins affecting sodium, potassium, and chloride channels.

Scorpions have survived successfully over millions of years without detectable changes in their morphology. Instead, they have developed an efficient alomonal machinery and a stinging device supporting their needs for prey and defense. They produce a large variety of polypeptidic toxins that bind and modulate ion channel conductance in excitable tissues. The binding site, mode of action, and chemical properties of many toxins have been studied extensively, but little is known about their genomic organization and diversity. Genes representing each of the major classes of Buthidae scorpion toxins, namely, "long" toxins, affecting sodium channels (alpha, depressant, and excitatory), and "short" toxins, affecting potassium and chloride channels, were isolated from a single scorpion segment and analyzed. Each toxin type was found to be encoded by a gene family. Regardless of toxin length, 3-D structure, and site of action, all genes contain A+T-rich introns that split, at a conserved location, an amino acid codon of the signal sequence. The introns vary in length and sequence but display identical boundaries, agree with the GT/AG splice junctions, and contain T-runs downstream of a putative branch point, 5'-TAAT-3'. Despite little sequence similarity among all toxin classes, the conserved gene organization, intron features, and common cysteine-stabilized alpha-helical (CSH) core connecting an alpha-helix to a three-stranded beta-sheet suggest, that they all evolved from an ancestral common progenitor. Furthermore, the vast diversity found among genomic copies, cDNAs, and their protein products for each toxin suggests an extensive evolutionary process of the scorpion "pharmaceutical factory," whose success is due, most likely, to the inherent permissiveness of the toxin exterior to structural alterations.

Membrane potential modulators: a thread of scarlet from plants to humans.

The preservation along evolution of specific core motifs in proteins of diverse functions and taxonomic origins pinpoints a possible developmental advantage at the structural level. Such a preservation was observed in a group of membrane potential modulators including plant gamma-thionins, scorpion toxins, insect and scorpion defensins, bee venom apamin and MCD peptide, snake sarafotoxins, and human endothelins. These substances are short polypeptides of various lengths and nonhomologous sequences that affect organisms of distant phyla. Despite the structural differences, comparative analysis reveals commonality at three levels: 1) effect on membrane potential; 2) a common cysteine-stabilized alpha-helical (CSH) motif; and 3) similar gene organization (except for insect defensins), i.e., an intron that splits a codon toward the end of the leader sequence. We thus propose that these modulators, divided into two groups differing in their CSH motif orientation, have either diverged from two independent ancestors or have evolved by gene diversification via exon shuffling and subsequent modifications. To enforce protein synthesis through the secretory pathway and enable disulfide bond formation and secretion, insertion sites downstream of preexisting leader sequences have been a prerequisite. What seems advantageous for evolution, may also be exploited in attempts to 'accelerate evolution' by protein design using the conserved CSH core as a suitable scaffold for reshaping molecular exteriors.

An excitatory scorpion toxin with a distinctive feature: an additional alpha helix at the C terminus and its implications for interaction with insect sodium channels.

BACKGROUND: Scorpion neurotoxins, which bind and modulate sodium channels, have been divided into two groups, the alpha and beta toxins, according to their activities. The beta-toxin class includes the groups of excitatory and depressant toxins, which differ in their mode of action and are highly specific against insects. The three-dimensional structures of several alpha and beta toxins have been determined at high resolution, but no detailed 3D structure of an excitatory toxin has been presented so far. RESULTS: The crystal structure of an anti-insect excitatory toxin from the scorpion Buthotus judaicus, Bj-xtrIT, has been determined at 2.1 A resolution and refined to an R factor of 0.209. The first 59 residues form a closely packed module, structurally similar to the conserved alpha and beta toxins ('long toxins') affecting sodium channels. The last 17 residues form a C-terminal extension not previously seen in scorpion toxins. It comprises a short alpha helix anchored to the N-terminal module by a disulfide bridge and is followed by a highly mobile stretch of seven residues, of which only four are seen in the electron-density map. This mobile peptide covers part of a conserved hydrophobic surface that is thought to be essential for interaction with the channel in several long toxins. CONCLUSIONS: Replacement of the last seven residues by a single glycine abolishes the activity of Bj-xtrIT, strongly suggesting that these residues are intimately involved in the interaction with the channel. Taken together with the partial shielding of the conserved hydrophobic surface and the proximity of the C terminus to an adjacent surface rich in charged residues, it seems likely that the bioactive surface of Bj-xtrIT is formed by residues surrounding the C terminus. The 3D structure and a recently developed expression system for Bj-xtrIT pave the way for identifying the structural determinants involved in the bioactivity and anti-insect specificity of excitatory toxins.

Baculovirus-mediated expression of a scorpion depressant toxin improves the insecticidal efficacy achieved with excitatory toxins.

The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti-insect selective toxins, was investigated. The ET50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET50 value of the wild-type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.

Identification of structural elements of a scorpion alpha-neurotoxin important for receptor site recognition.

alpha-Neurotoxins from scorpion venoms constitute the most studied group of modifiers of the voltage-sensitive sodium channels, and yet, their toxic site has not been characterized. We used an efficient bacterial expression system for modifying specific amino acid residues of the highly insecticidal alpha-neurotoxin LqhalphaIT from the scorpion Leiurus quinquestriatus hebraeus. Toxin variants modified at tight turns, the C-terminal region, and other structurally related regions were subjected to neuropharmacological and structural analyses. This approach highlighted both aromatic (Tyr10 and Phe17) and positively charged (Lys8, Arg18, Lys62, and Arg64) residues that (i) may interact directly with putative recognition points at the receptor site on the sodium channel; (ii) are important for the spatial arrangement of the toxin polypeptide; and (iii) contribute to the formation of an electrostatic potential that may be involved in biorecognition of the receptor site. The latter was supported by a suppressor mutation (E15A) that restored a detrimental effect caused by a K8D substitution. The feasibility of producing anti-insect scorpion neurotoxins with augmented toxicity was demonstrated by the substitution of the C-terminal arginine with histidine. Altogether, the present study provides for the first time an insight into the putative toxic surface of a scorpion neurotoxin affecting sodium channel gating.