Alon Harmelin

Artificial sweeteners induce glucose intolerance by altering the gut microbiota

Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.

MRI detection of transcriptional regulation of gene expression in transgenic mice.

Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R2) of water. Transgene-dependent changes in R2 were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.

Adenosine-to-inosine RNA editing shapes transcriptome diversity in primates

Human and chimpanzee genomes are almost identical, yet humans express higher brain capabilities. Deciphering the basis for this superiority is a long sought-after challenge. Adenosine-to-inosine (A-to-I) RNA editing is a widespread modification of the transcriptome. The editing level in humans is significantly higher compared with nonprimates, due to exceptional editing within the primate-specific Alu sequences, but the global editing level of nonhuman primates has not been studied so far. Here we report the sequencing of transcribed Alu sequences in humans, chimpanzees, and rhesus monkeys. We found that, on average, the editing level in the transcripts analyzed is higher in human brain compared with nonhuman primates, even where the genomic Alu structure is unmodified. Correlated editing is observed for pairs and triplets of specific adenosines along the Alu sequences. Moreover, new editable species-specific Alu insertions, subsequent to the human–chimpanzee split, are significantly enriched in genes related to neuronal functions and neurological diseases. The enhanced editing level in the human brain and the association with neuronal functions both hint at the possible contribution of A-to-I editing to the development of higher brain function. We show here that combinatorial editing is the most significant contributor to the transcriptome repertoire and suggest that Alu editing adapted by natural selection may therefore serve as an alternate information mechanism based on the binary A/I code.

Decreased hepcidin mRNA expression in thalassemic mice

Beta thalassemia major is a congenital haemolytic anaemia resulting from the lack of synthesis of the b-globin chain, a major component of haemoglobin A. In the absence of lifelong transfusions the disease is lethal. When adequate transfusion support is provided the patients suffer from iron overload that can lead to endocrine deficiencies, cirrhosis and cardiac failure that frequently lead to their demise. The iron overload results from ‘transfusional’ iron as well as from an inadequate high intestinal absorption despite high levels of body iron. The understanding of the deranged regulation of intestinal iron absorption is of utmost importance and may lead to rational therapy aimed at decreasing iron overload. Hepcidin, a recently identified anti-microbial peptide expressed in the liver was shown to play a role in conditions associated with both iron overload and iron deficiency. Hepcidin inhibits iron absorption in the proximal small bowel and the release of iron from macrophages. In healthy individuals as well as in murine models hepcidin expression is increased when iron stores are elevated and decreased as a result of iron deficiency anaemia and hypoxia (Nicolas et al, 2001; Park et al, 2001; Pigeon et al, 2001; Ganz, 2003). Thalassemia major represents a unique situation where anaemia, which is expected to decrease hepcidin expression, coexists with iron overload, which ought to increase it. Therefore, it was of interest to study which of these factors has an upper hand in hepcidin expression regulation. We used the recently described severe anaemia model of C57Bl/6 Hbb mice (May et al, 2002) that exhibit anaemia, abnormal red cell morphology, splenomegaly and develop spontaneous hepatic iron deposition (demonstrated by Gomori iron stain of liver tissue), and C57Bl/6 control mice in an attempt to understand the inadequately enhanced absorption of iron in thalassemia. RNA extracted from the liver of adult mice was analysed by quantifiable real time reverse transcription polymerase chain reaction (RT-PCR) with specific primers for the following iron metabolism-related genes: hepcidin, iron-regulated gene 1 (IREG1), neutral gelatinase-associated lipocalin (NGAL), haemochromatosis (HFE), transferrin receptors 1 and 2 (TfR1 and TfR2). The expression levels were normalized to b-actin. Interestingly, despite iron overload the expression levels of both hepcidin and TfR1 were significantly lower (0Æ38-fold) in the thalassemic mice. HFE was moderately decreased (0Æ7-fold), while NGAL was significantly increased (2Æ45-fold). TfR2 and IREG1 did not change significantly (Fig 1). This would suggest that iron overload is less dominant than anaemia in regulating hepcidin expression in the setting of the thalassemia major mouse model. The decreased expression of hepcidin may explain the increased absorption of iron in thalassemia. Recently, decreased expression of hepcidin was found in hereditary haemochromatosis in association with elevated levels of nontransferrin bound iron. The elevated expression of NGAL, an alternative iron delivery vehicle, supports the role of nontranferrin bound iron in the abnormal iron regulation in thalassemia. The decreased HFE expression level is similar to the finding in hereditary haemochromatosis (Bridle et al, 2003). The exact mechanism remains to be elucidated. These preliminary findings call for confirmation in human thalassemia patients. If supported, a new therapeutic approach to iron overload states such as thalassemia can be devised based on hepcidin administration or other interventions aimed at overcoming the inadequate response to iron overload associated with elevated levels of non-transferrin bound iron. Fig 1. Expression of iron regulatory genes in the liver of a mouse model of b-thalassemia. cDNA was prepared from the total RNA extracted from the liver of three C57B1/6 Hbb mice and three normal C57B1/6 wild-type mice. Gene expression of six iron regulatory genes was measured using specific primers for quantifiable reverse transcription polymerase chain reaction. Bars represent the average fold change in mRNA expression of each gene in C57B1/6 Hbb mice (black bars), when compared with control mRNA levels in C57B1/6 wild-type mice (grey bars). Each bar represents the average of 2–4 independent experiments, normalized to corresponding b-actin expression. correspondence

Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations

The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.

Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples

Estimating bacterial growth dynamics The pattern of sequencing read coverage of bacteria in metagenomic samples reflects the growth rate. This pattern is predictive of growth because bacterial genomes are circular, with a single origin of replication. So during growth, copies of the genome accumulate at the origin. Korem et al. use the ratio of copy number at the origin to the copy number at the terminus to detect the actively growing species in a microbiome (see the Perspective by Segre). They could spot the difference between virulent and avirulent strains, population diurnal oscillations, species that are growing in irritable bowel disease, and what happens when a hosts diet changes. Results were consistent in chemostats, in mice, and in human fecal samples. Science, this issue p. 1101; see also p. 1058 A new method provides a quantitative measure of the growth rate of multiple gut microbes in one go. [Also see Perspective by Segre] Metagenomic sequencing increased our understanding of the role of the microbiome in health and disease, yet it only provides a snapshot of a highly dynamic ecosystem. Here, we show that the pattern of metagenomic sequencing read coverage for different microbial genomes contains a single trough and a single peak, the latter coinciding with the bacterial origin of replication. Furthermore, the ratio of sequencing coverage between the peak and trough provides a quantitative measure of a species’ growth rate. We demonstrate this in vitro and in vivo, under different growth conditions, and in complex bacterial communities. For several bacterial species, peak-to-trough coverage ratios, but not relative abundances, correlated with the manifestation of inflammatory bowel disease and type II diabetes.

The anti‐inflammatory effects of 1,25‐dihydroxyvitamin D3 on Th2 cells in vivo are due in part to the control of integrin‐mediated T lymphocyte homing

The fat soluble vitamin D3 metabolite 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], and its nuclear receptor play an important role in regulating immune responses. While 1,25(OH)2D3 is known to inhibit transcription of cytokine genes that are required for Th1 differentiation or are products of differentiated Th1 cells, its role in regulating differentiation of Th2 cells is less clear. In this study, we show that 1,25(OH)2D3 has anti‐inflammatory effects in an in vivo Th2‐dependent asthma model. In addition, we demonstrate that 1,25(OH)2D3 down‐regulates the cytoskeleton rearrangement required for promoting integrin‐mediated adhesion of naive and effector CD4+ T cells. Finally, 1,25(OH)2D3 inhibits chemokine‐induced migration of naive cells and their homing to the lymph nodes. Thus, in addition to its regulation of cytokine transcription, 1,25(OH)2D3 regulates migration of cells and thus controls the skewing of various Th subsets in the secondary lymphoid organs and inhibits Th function at sites of inflammation.

Local photodynamic therapy (PDT) of rat C6 glioma xenografts with Pd-bacteriopheophorbide leads to decreased metastases and increase of animal cure compared with surgery

Photodynamic therapy (PDT), locally applied to solid C6 rat glioma tumors in the foot of CD1 nude mice, eradicated the primary tumor and also decreased the rate of groin and lung metastases. Pd‐Bacteriopheophorbide (Pd‐Bpheid), a novel photosensitizer synthesized in our laboratory, was used in our study. The primary lesion in the hind leg was treated by an i.v. injection of 5 mg/kg of Pd‐Bpheid and immediate illumination (650–800 nm, 360 J/cm2). This protocol and the surgical amputation of the leg were compared for local and metastasis responses. Following PDT, hemorrhage, inflammation with tumor necrosis and flattening were observed and histologically verified in the photodynamically treated tumor. Whereas local tumor control rates were up to 64% following PDT, in surgically treated animals, local tumor control was absolute. The rates of metastases in the groin and the lungs were at least 12‐fold lower in the photodynamically treated animals compared with untreated or surgery‐treated groups. The overall cure rates after PDT or surgery were 36% and 6%, respectively, at 8 weeks. These findings suggest that local PDT with Pd‐Bpheid, which acts primarily on the tumor vasculature, efficiently eradicates the solid C6 tumors. In addition, the local PDT of the primary lesion has beneficial therapeutic effects on remote C6 metastasis, which is not obtained with surgery. It is therefore suggested, that although surgery is highly efficient for the immediate removal of the primary tumor, it lacks such systemic, therapeutic effects on distant metastases. Pd‐Bpheid‐PDT may thus offer a potentially superior curative therapy for C6 glioma tumors in the limb by eradicating the target tumor and by reducing the rate of metastasis in the groin and lung, possibly due to innate immunity.

Persistent microbiome alterations modulate the rate of post-dieting weight regain

In tackling the obesity pandemic, considerable efforts are devoted to the development of effective weight reduction strategies, yet many dieting individuals fail to maintain a long-term weight reduction, and instead undergo excessive weight regain cycles. The mechanisms driving recurrent post-dieting obesity remain largely elusive. Here we identify an intestinal microbiome signature that persists after successful dieting of obese mice and contributes to faster weight regain and metabolic aberrations upon re-exposure to obesity-promoting conditions. Faecal transfer experiments show that the accelerated weight regain phenotype can be transmitted to germ-free mice. We develop a machine-learning algorithm that enables personalized microbiome-based prediction of the extent of post-dieting weight regain. Additionally, we find that the microbiome contributes to diminished post-dieting flavonoid levels and reduced energy expenditure, and demonstrate that flavonoid-based ‘post-biotic’ intervention ameliorates excessive secondary weight gain. Together, our data highlight a possible microbiome contribution to accelerated post-dieting weight regain, and suggest that microbiome-targeting approaches may help to diagnose and treat this common disorder.

Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a β cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.