Angeliki Karamitri

C/EBPβ Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression

cAMP-dependent protein kinase induction of PPARγ coactivator-1α (PGC-1α) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1α in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1α and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is cAMP-insensitive. A proximal 264-bp PGC-1α reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBPβ, but not C/EBPα and C/EBPδ, bound to the CRE on the PGC-1α promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBPβ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBPβ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBPβ rescued cAMP-inducible PGC-1α and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBPβ on the CRE in PGC-1α proximal promoter.

Combinatorial Transcription Factor Regulation of the Cyclic AMP-response Element on the Pgc-1α Promoter in White 3T3-L1 and Brown HIB-1B Preadipocytes

Cold stress in rodents increases the expression of UCP1 and PGC-1α in brown and white adipose tissue. We have previously reported that C/EBPβ specifically binds to the CRE on the proximal Pgc-1α promoter and increases forskolin-sensitive Pgc-1α and Ucp1 expression in white 3T3-L1 preadipocytes. Here we show that in mice exposed to a cold environment for 24 h, Pgc-1α, Ucp1, and C/ebpβ but not C/ebpα or C/ebpδ expression were increased in BAT. Conversely, expression of the C/EBP dominant negative Chop10 was increased in WAT but not BAT during cold exposure. Reacclimatization of cold-exposed mice to a warm environment for 24 h completely reversed these changes in gene expression. In HIB-1B, brown preadipocytes, forskolin increased expression of Pgc-1α, Ucp1, and C/ebpβ early in differentiation and inhibited Chop10 expression. Employing chromatin immunoprecipitation, we demonstrate that C/EBPβ, CREB, ATF-2, and CHOP10 are bound to the Pgc-1α proximal CRE, but CHOP10 does not bind in HIB-1B cell lysates. Forskolin stimulation and C/EBPβ overexpression in 3T3-L1 cells increased C/EBPβ and CREB but displaced ATF-2 and CHOP10 binding to the Pgc-1α proximal CRE. Overexpression of ATF-2 and CHOP10 in 3T3-L1 cells decreased Pgc-1α transcription. Knockdown of Chop10 in 3T3-L1 cells using siRNA increased Pgc-1α transcription, whereas siRNA against C/ebpβ in HIB-1B cells decreased Pgc-1α and Ucp1 expression. We conclude that the increased cAMP stimulation of Pgc-1α expression is regulated by the combinatorial effect of transcription factors acting at the CRE on the proximal Pgc-1α promoter.

Minireview: Toward the Establishment of a Link between Melatonin and Glucose Homeostasis: Association of Melatonin MT2 Receptor Variants with Type 2 Diabetes

The existence of interindividual variations in G protein-coupled receptor sequences has been recognized early on. Recent advances in large-scale exon sequencing techniques are expected to dramatically increase the number of variants identified in G protein-coupled receptors, giving rise to new challenges regarding their functional characterization. The current minireview will illustrate these challenges based on the MTNR1B gene, which encodes the melatonin MT2 receptor, for which exon sequencing revealed 40 rare nonsynonymous variants in the general population and in type 2 diabetes (T2D) cohorts. Functional characterization of these MT2 mutants revealed 14 mutants with loss of Gi protein activation that associate with increased risk of T2D development. This repertoire of disease-associated mutants is a rich source for structure-activity studies and will help to define the still poorly understood role of melatonin in glucose homeostasis and T2D development in humans. Defining the functional defects in carriers of rare MT2 mutations will help to provide personalized therapies to these patients in the future.

Cold-induced changes in gene expression in brown adipose tissue, white adipose tissue and liver

Cold exposure imposes a metabolic challenge to mammals that is met by a coordinated response in different tissues to prevent hypothermia. This study reports a transcriptomic analysis in brown adipose tissue (BAT), white adipose (WAT) and liver of mice in response to 24 h cold exposure at 8°C. Expression of 1895 genes were significantly (P<0.05) up- or down-regulated more than two fold by cold exposure in all tissues but only 5 of these genes were shared by all three tissues, and only 19, 14 and 134 genes were common between WAT and BAT, WAT and liver, and BAT and liver, respectively. We confirmed using qRT-PCR, the increased expression of a number of characteristic BAT genes during cold exposure. In both BAT and the liver, the most common direction of change in gene expression was suppression (496 genes in BAT and 590 genes in liver). Gene ontology analysis revealed for the first time significant (P<0.05) down regulation in response to cold, of genes involved in oxidoreductase activity, lipid metabolic processes and protease inhibitor activity, in both BAT and liver, but not WAT. The results reveal an unexpected importance of down regulation of cytochrome P450 gene expression and apolipoprotein, in both BAT and liver, but not WAT, in response to cold exposure. Pathway analysis suggests a model in which down regulation of the nuclear transcription factors HNF4α and PPARα in both BAT and liver may orchestrate the down regulation of genes involved in lipoprotein and steroid metabolism as well as Phase I enzymes belonging to the cytochrome P450 group in response to cold stress in mice. We propose that the response to cold stress involves decreased gene expression in a range of cellular processes in order to maximise pathways involved in heat production.

Design and validation of the first cell‐impermeant melatonin receptor agonist

The paradigm that GPCRs are able to prolong or initiate cellular signalling through intracellular receptors recently emerged. Melatonin binds to G protein‐coupled MT1 and MT2 receptors. In contrast to most other hormones targeting GPCRs, melatonin and its synthetic analogues are amphiphilic molecules easily penetrating into cells, but the existence of intracellular receptors is still unclear mainly due to a lack of appropriate tools.

The Difficult Journey from Genome-wide Association Studies to Pathophysiology: The Melatonin Receptor 1B (MT2) Paradigm

A recent study has investigated the relationship between the circadian neurohormone melatonin and glucose homeostasis and its implications for type 2 diabetes (T2D) risk (Tuomi et al., 2016). In particular, the authors have suggested that increased melatonin signaling is the mechanism by which the common T2D-associated SNP in MTNR1B (encoding melatonin MT2 receptor) identified in 2009 (Bouatia-Naji et al., 2009; Lyssenko et al., 2009) impacts glucose control.

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells

Acute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation.

Orphan GPR61, GPR62 and GPR135 receptors and the melatonin MT 2 receptor reciprocally modulate their signaling functions

Understanding the function of orphan G protein-coupled receptors (GPCRs), whose cognate ligand is unknown, is of major importance as GPCRs are privileged drug targets for many diseases. Recent phylogenetic studies classified three orphan receptors, GPR61, GPR62 and GPR135 among the melatonin receptor subfamily, but their capacity to bind melatonin and their biochemical functions are not well characterized yet. We show here that GPR61, GPR62 and GPR135 do not bind [3H]-melatonin nor 2-[125I]iodomelatonin and do not respond to melatonin in several signaling assays. In contrast, the three receptors show extensive spontaneous ligand-independent activities on the cAMP, inositol phosphate and ß-arrestin pathways with distinct pathway-specific profiles. Spontaneous ß-arrestin recruitment internalizes all three GPRs in the endosomal compartment. Co-expression of the melatonin binding MT2 receptor with GPR61, GPR62 or GPR135 has several consequences such as (i) the formation of receptor heteromers, (ii) the inhibition of melatonin-induced ß-arrestin2 recruitment to MT2 and (iii) the decrease of elevated cAMP levels upon melatonin stimulation in cells expressing spontaneously active GPR61 and GPR62. Collectively, these data show that GPR61, GPR62 and GPR135 are unable to bind melatonin, but show a reciprocal regulatory interaction with MT2 receptors.

Type 2 diabetes–associated variants of the MT2 melatonin receptor affect distinct modes of signaling

Connecting melatonin receptor variants to signaling differences reveals paths to type 2 diabetes therapy. Melatonin meets diabetes Some of the single-nucleotide polymorphisms associated with type 2 diabetes (T2D) occur in the gene encoding the melatonin receptor MT2, a G protein–coupled receptor (GPCR). Karamitri et al. measured the spontaneous and melatonin-stimulated signaling of 40 different MT2 variants. Computational analysis of these signaling profiles and assessment of genetic association data showed that those MT2 variants with defective melatonin-stimulated G protein signaling and reduced spontaneous β-arrestin recruitment were associated with the greatest risk for T2D. These data may aid in the development of specific treatments for T2D depending on the patient’s MT2 variant. Moreover, the experimental approach may be applied to assess the impact of other GPCR mutations on disease associations. Melatonin is produced during the night and regulates sleep and circadian rhythms. Loss-of-function variants in MTNR1B, which encodes the melatonin receptor MT2, a G protein–coupled receptor (GPCR), are associated with an increased risk of type 2 diabetes (T2D). To identify specific T2D-associated signaling pathway(s), we profiled the signaling output of 40 MT2 variants by monitoring spontaneous (ligand-independent) and melatonin-induced activation of multiple signaling effectors. Genetic association analysis showed that defects in the melatonin-induced activation of Gαi1 and Gαz proteins and in spontaneous β-arrestin2 recruitment to MT2 were the most statistically significantly associated with an increased T2D risk. Computational variant impact prediction by in silico evolutionary lineage analysis strongly correlated with the measured phenotypic effect of each variant, providing a predictive tool for future studies on GPCR variants. Together, this large-scale functional study provides an operational framework for the postgenomic analysis of the multiple GPCR variants present in the human population. The association of T2D risk with signaling pathway–specific defects opens avenues for pathway-specific personalized therapeutic intervention and reveals the potential relevance of MT2 function during the day, when melatonin is undetectable, but spontaneous activity of the receptor occurs.

Nocturnal activation of melatonin receptor type 1 signaling modulates diurnal insulin sensitivity via regulation of PI3K activity

Recent genetic studies have highlighted the potential involvement of melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2) in the pathogenesis of type 2 diabetes. Here, we report that mice lacking MT1 (MT1 KO) tend to accumulate more fat mass than WT mice and exhibit marked systemic insulin resistance. Additional experiments revealed that the main insulin signaling pathway affected by the loss of MT1 was the activation of phosphatidylinositol‐3‐kinase (PI3K). Transcripts of both catalytic and regulatory subunits of PI3K were strongly downregulated within MT1 KO mice. Moreover, the suppression of nocturnal melatonin levels within WT mice, by exposing mice to constant light, resulted in impaired PI3K activity and insulin resistance during the day, similar to what was observed in MT1 KO mice. Inversely, administration of melatonin to WT mice exposed to constant light was sufficient and necessary to restore insulin‐mediated PI3K activity and insulin sensitivity. Hence, our data demonstrate that the activation of MT1 signaling at night modulates insulin sensitivity during the day via the regulation of the PI3K transcription and activity. Lastly, we provide evidence that decreased expression of MTNR1A (MT1) in the liver of diabetic individuals is associated with poorly controlled diabetes.