DNA Sequence of Melanocortin 1-Receptor Gene in Coturnix Japonica: Correlation with Three E Locus Alleles, E, E+, Erh

The melanocortin 1-receptor (MC1-R) gene plays a key role in the expression of fur and feather color in mammals and birds by regulating the distribution of two melanin pigments: eumelanin (black/brown) and pheomelanin (red/yellow). MC1-R corresponds to the classical Extension (E) locus in mice, pigs, dogs, horses, and chickens. Three E locus alleles, the wild-type (e + ), brown (E), and redhead (e rh) have been identified in Japanese quail (Coturnix japonica). To determine if the quail E locus phenotypes were due to variation in the MC1-R gene, the coding region of the MC1-R gene was PCR amplified and DNA sequenced using genomic DNA isolated from individuals exhibiting the phenotypes of the three quail alleles. The DNA sequence comparison revealed two missense mutations that differentiated the brown from the wild-type and redhead quail. A single-base substitution resulted in a Val58Ile change, and another single-base substitution produced a Glu92Lys change in the brown quail. The redhead quail sequence carried a seven-base deletion extending from nucleotide position 682 to 688, resulting in a reading frame shift and premature termination of the MC1-R gene after amino acid position 231. The Glu92Lys change in the brown allele created a Msc I restriction fragment length polymorphism (RFLP). A PCR-Msc I RFLP test was developed and a direct correspondence between phenotype and genotype was found by testing the DNA of a population segregating for the brown and wild-type alleles. The DNA sequence and segregation data indicate that the quail E locus is homologous to the E locus identified in other birds and mammals

DNA sequence of melanocortin 1-receptor gene in Coturnix japonica: Correlation with three E locus alleles—E,e+, and erh

The melanocortin 1-receptor (MC1-R) gene plays a key role in the expression of fur and feather color in mammals and birds by regulating the distribution of two melanin pigments: eumelanin (black/brown) and pheomelanin (red/yellow). MC1-R corresponds to the classical Extension (E) locus in mice, pigs, dogs, horses, and chickens. Three E locus alleles, the wild-type (e+), brown (E), and redhead (erh) have been identified in Japanese quail (Coturnix japonica). To determine if the quail E locus phenotypes were due to variation in the MC1-R gene, the coding region of the MC1-R gene was PCR amplified and DNA sequenced using genomic DNA isolated from individuals exhibiting the phenotypes of the three quail alleles. The DNA sequence comparison revealed two missense mutations that differentiated the brown from the wild-type and redhead quail. A single-base substitution resulted in a Val58Ile change, and another single-base substitution produced a Glu92Lys change in the brown quail. The redhead quail sequence carried a seven-base deletion extending from nucleotide position 682 to 688, resulting in a reading frame shift and premature termination of the MC1-R gene after amino acid position 231. The Glu92Lys change in the brown allele created a Msc I restriction fragment length polymorphism (RFLP). A PCR-Msc I RFLP test was developed and a direct correspondence between phenotype and genotype was found by testing the DNA of a population segregating for the brown and wild-type alleles. The DNA sequence and segregation data indicate that the quail E locus is homologous to the E locus identified in other birds and mammals

Protective role of vitamin B6 (PLP) against DNA damage in Drosophila models of type 2 diabetes

Growing evidence shows that improper intake of vitamin B6 increases cancer risk and several studies indicate that diabetic patients have a higher risk of developing tumors. We previously demonstrated that in Drosophila the deficiency of Pyridoxal 5' phosphate (PLP), the active form of vitamin B6, causes chromosome aberrations (CABs), one of cancer prerequisites, and increases hemolymph glucose content. Starting from these data we asked if it was possible to provide a link between the aforementioned studies. Thus, we tested the effect of low PLP levels on DNA integrity in diabetic cells. To this aim we generated two Drosophila models of type 2 diabetes, the first by impairing insulin signaling and the second by rearing flies in high sugar diet. We showed that glucose treatment induced CABs in diabetic individuals but not in controls. More interestingly, PLP deficiency caused high frequencies of CABs in both diabetic models demonstrating that hyperglycemia, combined to reduced PLP level, impairs DNA integrity. PLP-depleted diabetic cells accumulated Advanced Glycation End products (AGEs) that largely contribute to CABs as α-lipoic acid, an AGE inhibitor, rescued not only AGEs but also CABs. These data, extrapolated to humans, indicate that low PLP levels, impacting on DNA integrity, may be considered one of the possible links between diabetes and cancer

Inter-organelle ER-endolysosomal contact sites in metabolism and disease across evolution

Since their initial observation, contact sites formed between different organelles have transitioned from ignored curiosities to recognized centers for the exchange of metabolites and lipids. Contact formed between the ER and endomembrane system (eg. the plasma membrane, endosomes, and lysosomes) is of particular biomedical interest, as it governs aspects of lipid metabolism, organelle identity, and cell signaling. Here, we review the field of ER-endolysosomal communication from the perspective of three model systems: budding yeast, the fruit fly D. melanogaster, and mammals. From this broad perspective, inter-organelle communication displays a consistent role in metabolic regulation that was differentially tuned during the development of complex metazoan life. We also examine the current state of understanding of lipid exchange between organelles, and discuss molecular mechanisms by which this occurs

Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism

Circulating carbohydrates are an essential energy source, perturbations in which are pathognomonic of various diseases, diabetes being the most prevalent. Yet many of the genes underlying diabetes and its characteristic hyperglycaemia remain elusive. Here we use physiological and genetic interrogations in D. melanogaster to uncover the ‘glucome', the complete set of genes involved in glucose regulation in flies. Partial genomic screens of ∼1,000 genes yield ∼160 hyperglycaemia ‘flyabetes' candidates that we classify using fat body- and muscle-specific knockdown and biochemical assays. The results highlight the minor glucose fraction as a physiological indicator of metabolism in Drosophila. The hits uncovered in our screen may have conserved functions in mammalian glucose homeostasis, as heterozygous and homozygous mutants of Ck1alpha in the murine adipose lineage, develop diabetes. Our findings demonstrate that glucose has a role in fly biology and that genetic screenings carried out in flies may increase our understanding of mammalian pathophysiology

Drosophila snazarus regulates a lipid droplet population at plasma membrane-droplet contacts in adipocytes

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes, we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk

An ancestral role for the mitochondrial pyruvate carrier in glucose-stimulated insulin secretion

Objective: Transport of pyruvate into the mitochondrial matrix by the Mitochondrial Pyruvate Carrier (MPC) is an important and rate-limiting step in its metabolism. In pancreatic β-cells, mitochondrial pyruvate metabolism is thought to be important for glucose sensing and glucose-stimulated insulin secretion. Methods: To evaluate the role that the MPC plays in maintaining systemic glucose homeostasis, we used genetically-engineered Drosophila and mice with loss of MPC activity in insulin-producing cells. Results: In both species, MPC deficiency results in elevated blood sugar concentrations and glucose intolerance accompanied by impaired glucose-stimulated insulin secretion. In mouse islets, β-cell MPC-deficiency resulted in decreased respiration with glucose, ATP-sensitive potassium (KATP) channel hyperactivity, and impaired insulin release. Moreover, treatment of pancreas-specific MPC knockout mice with glibenclamide, a sulfonylurea KATP channel inhibitor, improved defects in islet insulin secretion and abnormalities in glucose homeostasis in vivo. Finally, using a recently-developed biosensor for MPC activity, we show that the MPC is rapidly stimulated by glucose treatment in INS-1 insulinoma cells suggesting that glucose sensing is coupled to mitochondrial pyruvate carrier activity. Conclusions: Altogether, these studies suggest that the MPC plays an important and ancestral role in insulin-secreting cells in mediating glucose sensing, regulating insulin secretion, and controlling systemic glycemia. Keywords: Stimulus-coupled secretion, Insulin, β-Cell, Diabetes, Pyruvate, Mitochondria, Drosophil