Increased FGF21 in brown adipose tissue of tyrosine hydroxylase heterozygous mice: implications for cold adaptation.

Tyrosine hydroxylase (TH) catalyzes the first step in catecholamines synthesis. We studied the impact of reduced TH in brown adipose tissue (BAT) activation. In adult heterozygous (Th+/- ) mice, dopamine and noradrenaline (NA) content in BAT decreased after cold exposure. This reduced catecholaminergic response did not impair cold adaptation, because these mice induced uncoupling protein 1 (UCP-1) and maintained BAT temperature to a similar extent than controls (Th+/+ ). Possible compensatory mechanisms implicated were studied. Prdm16 and Fgf21 expression, key genes in BAT activation, were elevated in Th+/- mice at thermoneutrality from day 18.5 of embryonic life. Likewise, plasma FGF21 and liver Fgf21 mRNA were increased. Analysis of endoplasmic reticulum (ER) stress, a process that triggers elevations in FGF21, showed higher phospho-IRE1, phospho-JNK, and CHOP in BAT of Th+/- mice at thermoneutrality. Also, increased lipolysis in BAT of cold-exposure Th+/- mice was demonstrated by increased phosphorylation of hormone-sensitive lipase (HSL), as well as diacylglycerol (DAG) and FFA content. Overall, these results indicate that the mild effects of Th haploinsufficiency on BAT function are likely due to compensatory mechanisms involving elevations in Fgf21 and Prdm16 and through adaptive changes in the lipid profile.

Tyrosine hydroxylase haploinsufficiency prevents age-associated arterial pressure elevation and increases half-life in mice.

Catecholamines are essential for the maintenance of physiological homeostasis under basal and stress conditions. We aim to determine the impact of deletion of a single allele of the tyrosine hydroxylase (Th) gene might have on aging arterial pressure and life-span. We found that Th haploinsufficiency prevents age-associated increase of arterial pressure (AP) in mature adult mice, and it results in the extension of the half-life of Th-heterozygous (TH-HET) mice respect to their wild-type (WT) littermates. Heart performance was similar in both genotypes. To further investigate the lack of increase in AP with age in TH-HET mice, we measured the AP response to intra-peritoneal administration of substances involved in AP regulation. The response to acetylcholine and the basal sympathetic tone were similar in both genotypes, while norepinephrine had a greater pressor effect in TH-HET mice, which correlated with altered adrenoreceptor expression in blood vessels and the heart. Furthermore, sympatho-adrenomedular response to stress was attenuated in TH-HET mice. Plasma catecholamine levels and urine glucose increased markedly in WT but not in TH-HET mice after stress. Our results showed that TH-HET mice are resistant to age-associated hypertension, present a reduction in the sympathetic response to stress and display an extended half-life.

Non-neural tyrosine hydroxylase, via modulation of endocrine pancreatic precursors, is required for normal development of beta cells in the mouse pancreas.

AIMS/HYPOTHESIS: Apart from transcription factors, little is known about the molecules that modulate the proliferation and differentiation of pancreatic endocrine cells. The early expression of tyrosine hydroxylase (TH) in a subset of glucagon(+) cells led us to investigate whether catecholamines have a role in beta cell development. METHODS: We studied the immunohistochemical characteristics of TH-expressing cells in wild-type (Th (+/+) ) mice during early pancreas development, and analysed the endocrine pancreas phenotype of TH-deficient (Th (-/-) ) mice. We also studied the effect of dopamine addition and TH-inhibition on insulin-producing cells in explant cultures. RESULTS: In the mouse pancreas at embryonic day (E)12.5-E13.5, the ∼10% of early glucagon(+) cells that co-expressed TH rarely proliferated and did not express the precursor marker neurogenin 3 at E13.5. The number of insulin(+) cells in the Th (-/-) embryonic pancreas was decreased as compared with wild-type embryos at E13.5. While no changes in pancreatic and duodenal homeobox 1 (PDX1)(+)-progenitor cell number were observed between groups at E12.5, the number of neurogenin 3 and NK2 homeobox 2 (NKX2.2)-expressing cells was reduced in Th (-/-) embryonic pancreas, an effect that occurred in parallel with increased expression of the transcriptional repressor Hes1. The potential role of dopamine as a pro-beta cell stimulus was tested by treating pancreas explants with this catecholamine, which resulted in an increase in total insulin content and insulin(+) cells relative to control explants. CONCLUSIONS/INTERPRETATION: A non-neural catecholaminergic pathway appears to modulate the pancreatic endocrine precursor and insulin producing cell neogenesis. This finding may have important implications for approaches seeking to promote the generation of beta cells to treat diabetes.

Early neural cell death is an extensive, dynamic process in the embryonic chick and mouse retina.

Orchestrated proliferation, differentiation, and cell death contribute to the generation of the complex cytoarchitecture of the central nervous system, including that of the neuroretina. However, few studies have comprehensively compared the spatiotemporal patterns of these 3 processes, or their relative magnitudes. We performed a parallel study in embryonic chick and mouse retinas, focusing on the period during which the first neurons, the retinal ganglion cells (RGCs), are generated. The combination of in vivo BrdU incorporation, immunolabeling of retinal whole mounts for BrdU and for the neuronal markers Islet1/2 and ß III-tubulin, and TUNEL allowed for precise cell scoring and determination the spatiotemporal patterns of cell proliferation, differentiation, and death. As predicted, proliferation preceded differentiation. Cell death and differentiation overlapped to a considerable extent, although the magnitude of cell death exceeded that of neuronal differentiation. Precise quantification of the population of recently born RGCs, identified by BrdU and ß III-tubulin double labeling, combined with cell death inhibition using a pan-caspase inhibitor, revealed that apoptosis decreased this population by half shortly after birth. Taken together, our findings provide important insight into the relevance of cell death in neurogenesis.

The T-box brain 1 (Tbr1) transcription factor inhibits astrocyte formation in the olfactory bulb and regulates neural stem cell fate.

The T-box brain 1 (Tbr1) gene encodes a transcription factor necessary for the maintenance and/or differentiation of glutamatergic cells in the olfactory bulb (OB) and cortex, although its precise function in the development of glutamatergic neurons is not known. Furthermore, Tbr1 has not been reported to regulate the formation of glial cells. We show that Tbr1 is expressed during the initial stages in the generation of glutamatergic mitral neurons from dividing progenitors in the E12.5 mouse OB. Retroviral-mediated overexpression of Tbr1 in cultured embryonic and adult OB stem cells (OBSC) produces a marked increase in the number of TuJ1(+) neurons (including VGLUT1(+) glutamatergic and GABA(+) neurons) and O4(+) oligodendrocytes. Moreover, transduction of Tbr1 inhibits the production of GFAP(+) astrocytes from both cultured OBSC and dividing progenitor cells in vivo. These results show that the expression of Tbr1 in neural stem and progenitor cells prevents them from following an astrocyte fate during OB development. Our findings suggest that the transduction of Tbr1 into neural stem cells could be useful to increase the production of neurons and oligodendrocytes in studies of neuroregeneration.

Evolution of the insulin receptor family and receptor isoform expression in vertebrates.

The molecular phylogeny of the vertebrate insulin receptor (IR) family was reconstructed under maximum likelihood (ML) to establish homologous relationships among its members. A sister group relationship between the orphan insulin-related receptor (IRR) and the insulin-like growth factor 1 receptor (IGF1R) to the exclusion of the IR obtained maximal bootstrap support. Although both IR and IGF1R were identified in all vertebrates, IRR could not be found in any teleost fish. The ancestral character states at each position of the receptor molecule were inferred for IR, IRR + IGF1R, and all 3 paralogous groups based on the recovered phylogeny using ML in order to determine those residues that could be important for the specific function of IR. For 18 residues, ancestral character state of IR was significantly distinct (probability >0.95) with respect to the corresponding inferred ancestral character states both of IRR + IGF1R and of all 3 vertebrate paralogs. Most of these IR distinct (shared derived) residues were located on the extracellular portion of the receptor (because this portion is larger and the rate of generation of IR shared derived sites is uniform along the receptor), suggesting that functional diversification during the evolutionary history of the family was largely generated modifying ligand affinity rather than signal transduction at the tyrosine kinase domain. In addition, 2 residues at positions 436 and 1095 of the human IR sequence were identified as radical cluster-specific sites in IRR + IGF1R. Both Ir and Irr have an extra exon (namely exon 11) with respect to Igf1r. We used the molecular phylogeny to infer the evolution of this additional exon. The Irr exon 11 can be traced back to amphibians, whereas we show that presence and alternative splicing of Ir exon 11 seems to be restricted exclusively to mammals. The highly divergent sequence of both exons and the reconstructed phylogeny of the vertebrate IR family strongly indicate that both exons were acquired independently by each paralog.

IGF-I promotes neuronal migration and positioning in the olfactory bulb and the exit of neuroblasts from the subventricular zone.

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I (-/-) mice. In these animals, Tbr1(+)-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I (-/-) mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I (-/-) mice. Significantly, the positioning of Tbr1(+)-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I (-/-) mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I (-/-) was similar to that from Igf-I (+/+) explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.

Differential effects of metformin glycinate and hydrochloride in glucose production, AMPK phosphorylation and insulin sensitivity in hepatocytes from non-diabetic and diabetic mice.

The liver is a main target tissue of the biguanide metformin which activates AMP-activated protein kinase (AMPK). We previously reported that administration of metformin glycinate showed a greater decrease of glycated hemoglobin A1c than a placebo in patients with type 2 diabetes mellitus (T2DM). In this study, we compared the effects of metformin hydrochloride, the oral antidiabetic drug of first choice, with those of metformin glycinate in hepatocytes from non-diabetic and diabetic mice and humans. Both formulations were equally potent regard to the reduction of basal and glucagon-induced glucose production and mRNA levels of gluconeogenic enzymes (Pck1 and G6pc) in hepatocytes from C57/Bl6 mice and humans. On the contrary, phosphorylation of AMPK and its substrate acetyl CoA carboxylase (ACC) was faster in hepatocytes treated with metformin glycinate. Likewise, we found stronger reduction in hepatocytes from obese/diabetic db/db mice of glucagon-induced glucose output and more sustained AMPK phosphorylation after treatment with metformin glycinate. Importantly, insulin sensitization regarding phosphorylation of AKT (Ser473) was enhanced in hepatocytes from db/db mice or humans pretreated with metformin glycinate. In conclusion, our data indicate that metformin glycinate may be an alternative therapy against insulin resistance during obesity and/or T2DM.

The regulated expression of chimeric tyrosine hydroxylase-insulin transcripts during early development.

Biological complexity does not appear to be simply correlated with gene number but rather other mechanisms contribute to the morphological and functional diversity across phyla. Such mechanisms regulate different transcriptional, translational and post-translational processes and include the recently identified transcription induced chimerism (TIC). We have found two novel chimeric transcripts in the chick and quail that result from the fusion of tyrosine hydroxylase (TH) and insulin into a single mature transcript. The th and insulin genes are located in tandem and they are generally transcribed independently. However, it appears that two chimeric transcripts containing exons from both the genes can also be produced in a regulated manner. The TH-INS1 and TH-INS2 chimeras differ in their insulin gene content, and they encode two novel isoforms of the TH protein with markedly reduced functionality when compared with the canonical TH. In addition, the TH-INS1 chimeric mRNA generates a small amount of insulin. We propose that TIC is an additional mechanism that can be employed to further regulate TH and insulin expression according to the specific needs of developing vertebrates.

The Prohormone Proinsulin as a Neuroprotective Factor: Past History and Future Prospects.

Proinsulin was first identified as the primary translation product of the insulin gene in Donald Steiner's laboratory in 1967, and was the first prohormone to be isolated and sequenced. While its role as an insulin precursor has been extensively studied in the field of endocrinology, the bioactivity of the proinsulin molecule itself has received much less attention. Insulin binds to isoforms A and B of the insulin receptor (IR) with high affinity. Proinsulin, in contrast, binds with high affinity only to IR-A, which is present in the nervous system, among other tissues and elicits antiapoptotic and neuroprotective effects in the developing and postnatal nervous system. Proinsulin specifically exerts neuroprotection in the degenerating retina in mouse and rat models of retinitis pigmentosa (RP), delaying photoreceptor and vision loss after local administration in the eye or systemic (intramuscular) administration of an adeno-associated viral (AAV) vector that induces constitutive proinsulin release. AAV-mediated proinsulin expression also decreases the expression of neuroinflammation markers in the hippocampus and sustains cognitive performance in a mouse model of precocious brain senescence. We have therefore proposed that proinsulin should be considered a functionally distinct member of the insulin superfamily. Here, we briefly review the legacy of Steiner's research, the neural expression of proinsulin, and the tissue expression patterns and functional characteristics of IR-A. We discuss the neuroprotective activity of proinsulin and its potential as a therapeutic tool in neurodegenerative conditions of the central nervous system, particularly in retinal dystrophies.