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1.
Endocrinology ; 163(1)2022 01 01.
Article in English | MEDLINE | ID: mdl-34919671

ABSTRACT

MicroRNAs (miRNAs) expressed in the hypothalamus are capable of regulating energy balance and peripheral metabolism by inhibiting translation of target messenger RNAs (mRNAs). Hypothalamic insulin resistance is known to precede that in the periphery, thus a critical unanswered question is whether central insulin resistance creates a specific hypothalamic miRNA signature that can be identified and targeted. Here we show that miR-1983, a unique miRNA, is upregulated in vitro in 2 insulin-resistant immortalized hypothalamic neuronal neuropeptide Y-expressing models, and in vivo in hyperinsulinemic mice, with a concomitant decrease of insulin receptor ß subunit protein, a target of miR-1983. Importantly, we demonstrate that miR-1983 is detectable in human blood serum and that its levels significantly correlate with blood insulin and the homeostatic model assessment of insulin resistance. Levels of miR-1983 are normalized with metformin exposure in mouse hypothalamic neuronal cell culture. Our findings provide evidence for miR-1983 as a unique biomarker of cellular insulin resistance, and a potential therapeutic target for prevention of human metabolic disease.


Subject(s)
Hypothalamus/metabolism , Insulin/pharmacology , Metformin/pharmacology , MicroRNAs/genetics , Receptor, Insulin/genetics , Adult , Animals , Cell Line , Cells, Cultured , Female , Gene Expression Profiling/methods , Gene Expression Regulation/drug effects , Humans , Hypoglycemic Agents/pharmacology , Hypothalamus/cytology , Insulin/blood , Insulin/metabolism , Insulin Resistance/genetics , Male , Mice , MicroRNAs/blood , Middle Aged , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Obesity/blood , Obesity/genetics , Obesity/metabolism , Receptor, Insulin/metabolism
2.
J Endocrinol ; 220(1): 13-24, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24134870

ABSTRACT

POMC neurons play a central role in the maintenance of whole-body energy homeostasis. This balance requires proper regulation of POMC neurons by metabolic hormones, such as insulin. However, the heterogeneous cellular population of the intact hypothalamus presents challenges for examining the molecular mechanisms underlying the potent anorexigenic effects of POMC neurons, and there is currently a complete lack of mature POMC neuronal cell models for study. To this end, we have generated novel, immortalized, adult-derived POMC-expressing/α-MSH-secreting cell models, mHypoA-POMC/GFP lines 1-4, representing the fluorescence-activated cell-sorted POMC population from primary POMC-eGFP mouse hypothalamus. The presence of Pomc mRNA in these cell lines was confirmed, and α-MSH was detected via immunofluorescence. α-MSH secretion in the mHypoA-POMC/GFP-1 was found to increase in response to 10  ng/ml ciliary neurotrophic factor (CNTF) or 10  nM insulin as determined by enzyme immunoassay. Further experiments using the mHypoA-POMC/GFP-1 cell line revealed that 10  ng/ml CNTF increases Pomc mRNA at 1 and 2  h after treatment, whereas insulin elicited an increase in Pomc mRNA level and decreases in insulin receptor (Insr (Ir)) mRNA level at 4  h. Furthermore, the activation of IR-mediated downstream second messengers was examined by western blot analysis, following the induction of cellular insulin resistance, which resulted in a loss of insulin-mediated regulation of Pomc and Ir mRNAs. The development of these immortalized neurons will be invaluable for the elucidation of the cellular and molecular mechanisms that underlie POMC neuronal function under normal and perturbed physiological conditions.


Subject(s)
Insulin/pharmacology , Neurons/metabolism , Pro-Opiomelanocortin/genetics , Signal Transduction/drug effects , Animals , Blotting, Western , Cell Line, Transformed , Ciliary Neurotrophic Factor/pharmacology , Drug Resistance , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hypoglycemic Agents/pharmacology , Hypothalamus/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons/cytology , Phosphorylation/drug effects , Pro-Opiomelanocortin/metabolism , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , alpha-MSH/metabolism
3.
Mol Endocrinol ; 27(6): 990-1003, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23579487

ABSTRACT

Central resistance to the actions of insulin and leptin is associated with the onset of obesity and type 2 diabetes mellitus, whereas leptin and insulin signaling is essential for both glucose and energy homeostasis. Although it is known that leptin resistance can lead to attenuated insulin signaling, whether insulin resistance can lead to or exacerbate leptin resistance is unknown. To investigate the molecular events underlying crosstalk between these signaling pathways, immortalized hypothalamic neuronal models, rHypoE-19 and mHypoA-2/10, were used. Prolonged insulin exposure was used to induce cellular insulin resistance, and thereafter leptin-mediated regulation of signal transduction and gene expression was assessed. Leptin directly repressed agouti-related peptide mRNA levels but induced urocortin-2, insulin receptor substrate (IRS)-1, IRS2, and IR transcription, through leptin-mediated phosphatidylinositol 3-kinase/Akt activation. Neuronal insulin resistance, as assessed by attenuated Akt phosphorylation, blocked leptin-mediated signal transduction and agouti-related peptide, urocortin-2, IRS1, IRS2, and insulin receptor synthesis. Insulin resistance caused a substantial decrease in insulin receptor protein levels, forkhead box protein 1 phosphorylation, and an increase in suppressor of cytokine signaling 3 protein levels. Cellular insulin resistance may cause or exacerbate neuronal leptin resistance and, by extension, obesity. It is essential to unravel the effects of neuronal insulin resistance given that both peripheral, as well as the less widely studied central insulin resistance, may contribute to the development of metabolic, reproductive, and cardiovascular disorders. This study provides improved understanding of the complex cellular crosstalk between insulin-leptin signal transduction that is disrupted during neuronal insulin resistance.


Subject(s)
Insulin Resistance , Leptin/physiology , Signal Transduction , Transcription, Genetic , Androstadienes/pharmacology , Animals , Cells, Cultured , Chromones/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Hypothalamus/cytology , Insulin/physiology , Janus Kinase 2/metabolism , Mice , Morpholines/pharmacology , Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Rats , STAT3 Transcription Factor/metabolism , Wortmannin
4.
Endocrinology ; 153(5): 2208-22, 2012 May.
Article in English | MEDLINE | ID: mdl-22334721

ABSTRACT

Exendin-4, a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, is a potential regulator of feeding behavior through its ability to inhibit gastric emptying, reduce food intake, and induce satiety. GLP-1R activation by exendin-4 induces anorexia; however, the specific populations of neuropeptidergic neurons activated by exendin-4 within the hypothalamus, the central regulator of energy homeostasis, remain unclear. This study determines whether exendin-4 regulates hypothalamic neuropeptide expression and explores the signaling mechanisms involved. The distribution and quantity of exendin-4-induced c-Fos immunoreactivity were evaluated to determine activation of α-melanocyte-stimulating hormone/proopiomelanocortin, neuropeptide Y, neurotensin (NT), and ghrelin neurons in hypothalamic nuclei during exendin-4-induced anorexia in mice. Additionally, exendin-4 action on NT and ghrelin transcript regulation was examined in immortalized hypothalamic neurons. With anorexia induced by intracerebroventricular exendin-4, α-melanocyte-stimulating hormone/proopiomelanocortin and neuropeptide Y neurons were activated in the arcuate nucleus, with simultaneous activation of NT-expressing neurons in the paraventricular nucleus, and ghrelin-expressing neurons in the arcuate nucleus, paraventricular nucleus, and periventricular hypothalamus, suggesting that neurons in one or more of these areas mediate the anorexic action of exendin-4. In the hypothalamic neuronal cell models, exendin-4 increased cAMP, cAMP response element-binding protein/activating transcription factor-1 and c-Fos activation, and via a protein kinase A-dependent mechanism regulated NT and ghrelin mRNA expression, indicating that these neuropeptides may serve as downstream mediators of exendin-4 action. These findings provide a previously unrecognized link between central GLP-1R activation by exendin-4 and the regulation of hypothalamic NT and ghrelin. Further understanding of this central GLP-1R activation may lead to safe and effective therapeutics for the treatment of metabolic disorders.


Subject(s)
Hypothalamus/drug effects , Neurons/drug effects , Peptides/pharmacology , Receptors, Glucagon/agonists , Venoms/pharmacology , Animals , Eating/drug effects , Eating/physiology , Exenatide , Feeding Behavior/drug effects , Feeding Behavior/physiology , Ghrelin/metabolism , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor , Hypothalamus/metabolism , Mice , Neurons/metabolism , Neuropeptide Y/metabolism , Neurotensin/metabolism , Pro-Opiomelanocortin/metabolism , Proto-Oncogene Proteins c-fos/metabolism , alpha-MSH/metabolism
5.
PLoS Genet ; 7(11): e1002377, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22125496

ABSTRACT

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.


Subject(s)
Cell Membrane/genetics , Epistasis, Genetic/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Membrane Proteins/genetics , Microtubule-Associated Proteins/genetics , Culture Media , Drug Resistance/genetics , Escherichia coli/growth & development , Gene Expression Regulation, Bacterial , Gene-Environment Interaction , Membrane Proteins/metabolism , Metabolic Networks and Pathways/genetics , Microscopy, Electron , Microtubule-Associated Proteins/metabolism , Molecular Sequence Annotation , Oligonucleotide Array Sequence Analysis
6.
Am J Physiol Regul Integr Comp Physiol ; 300(5): R1030-52, 2011 May.
Article in English | MEDLINE | ID: mdl-21248304

ABSTRACT

The hypothalamus is a vital part of the central nervous system: it harbors control systems implicated in regulation of a wide range of homeostatic processes, including energy balance and reproduction. Structurally, the hypothalamus is a complex neuroendocrine tissue composed of a multitude of unique neuronal cell types that express a number of neuromodulators, including hormones, classical neurotransmitters, and specific neuropeptides that play a critical role in mediating hypothalamic function. However, neuropeptide and receptor gene expression, second messenger activation, and electrophysiological and secretory properties of these hypothalamic neurons are not yet fully defined, primarily because the heterogeneity and complex neuronal architecture of the neuroendocrine hypothalamus make such studies challenging to perform in vivo. To circumvent this problem, our research group recently generated embryonic- and adult-derived hypothalamic neuronal cell models by utilizing the novel molecular techniques of ciliary neurotrophic factor-induced neurogenesis and SV40 T antigen transfer to primary hypothalamic neuronal cell cultures. Significant research with these cell lines has demonstrated their value as a potential tool for use in molecular genetic analysis of hypothalamic neuronal function. Insights gained from hypothalamic immortalized cells used in conjunction with in vivo models will enhance our understanding of hypothalamic functions such as neurogenesis, neuronal plasticity, glucose sensing, energy homeostasis, circadian rhythms, and reproduction. This review discusses the generation and use of hypothalamic cell models to study mechanisms underlying the function of individual hypothalamic neurons and to gain a more complete understanding of the overall physiology of the hypothalamus.


Subject(s)
Cell Culture Techniques , Hypothalamus/physiology , Neurons/physiology , Animals , Cell Line, Transformed , Energy Metabolism , Gene Expression Regulation , Glucose/metabolism , Homeostasis , Humans , Hypothalamus/cytology , Neurogenesis , Neuronal Plasticity , Neuropeptides/metabolism , Phenotype , Receptors, Neuropeptide/metabolism , Reproduction , Signal Transduction
7.
PLoS Biol ; 7(4): e96, 2009 Apr 28.
Article in English | MEDLINE | ID: mdl-19402753

ABSTRACT

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Genome, Bacterial , Proteome/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Multiprotein Complexes/genetics , Protein Interaction Mapping/methods
8.
Nat Methods ; 5(9): 789-95, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18677321

ABSTRACT

Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.


Subject(s)
Escherichia coli/genetics , Oligonucleotide Array Sequence Analysis , Conjugation, Genetic , Genome, Bacterial , Mutation
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