ABSTRACT
The maternal perinatal environment modulates brain formation, and altered maternal nutrition has been linked to the development of metabolic and psychiatric disorders in the offspring. Here, we showed that maternal high-fat diet (HFD) feeding during lactation in mice elicits long-lasting changes in gene expression in the offspring's dopaminergic circuitry. This translated into silencing of dopaminergic midbrain neurons, reduced connectivity to their downstream targets, and reduced stimulus-evoked dopamine (DA) release in the striatum. Despite the attenuated activity of DA midbrain neurons, offspring from mothers exposed to HFD feeding exhibited a sexually dimorphic expression of DA-related phenotypes, i.e., hyperlocomotion in males and increased intake of palatable food and sucrose in females. These phenotypes arose from concomitantly increased spontaneous activity of D1 medium spiny neurons (MSNs) and profoundly decreased D2 MSN projections. Overall, we have unraveled a fundamental restructuring of dopaminergic circuitries upon time-restricted altered maternal nutrition to induce persistent behavioral changes in the offspring.
Subject(s)
Diet, High-Fat/adverse effects , Dopamine/metabolism , Lactation , Maternal Exposure/adverse effects , Mesencephalon/metabolism , Animals , Female , Male , Mesencephalon/pathology , MiceABSTRACT
Unlimited access to calorie-dense, palatable food is a hallmark of Western societies and substantially contributes to the worldwide rise of metabolic disorders. In addition to promoting overconsumption, palatable diets dampen daily intake patterns, further augmenting metabolic disruption. We developed a paradigm to reveal differential timing in the regulation of food intake behavior in mice. While homeostatic intake peaks in the active phase, conditioned place preference and choice experiments show an increased sensitivity to overeating on palatable food during the rest phase. This hedonic appetite rhythm is driven by endogenous circadian clocks in dopaminergic neurons of the ventral tegmental area (VTA). Mice with disrupted clock function in the VTA lose their hedonic overconsumption rhythms without affecting homeostatic intake. These findings assign a functional role of VTA clocks in modulating palatable feeding behaviors and identify a potential therapeutic route to counteract hyperphagy in an obesogenic environment.
Subject(s)
Circadian Rhythm , Dopaminergic Neurons/physiology , Feeding Behavior , Ventral Tegmental Area/physiology , Animals , Appetite , Behavior, Animal , Choice Behavior , Homeostasis , Male , Mice , Mice, Inbred C57BL , Obesity/metabolism , OscillometryABSTRACT
Obesity promotes the development of insulin resistance and increases the incidence of colitis-associated cancer (CAC), but whether a blunted insulin action specifically in intestinal epithelial cells (IECs) affects CAC is unknown. Here, we show that obesity impairs insulin sensitivity in IECs and that mice with IEC-specific inactivation of the insulin and IGF1 receptors exhibit enhanced CAC development as a consequence of impaired restoration of gut barrier function. Blunted insulin signalling retains the transcription factor FOXO1 in the nucleus to inhibit expression of Dsc3, thereby impairing desmosome formation and epithelial integrity. Both IEC-specific nuclear FoxO1ADA expression and IEC-specific Dsc3 inactivation recapitulate the impaired intestinal integrity and increased CAC burden. Spontaneous colonic tumour formation and compromised intestinal integrity are also observed upon IEC-specific coexpression of FoxO1ADA and a stable Myc variant, thus suggesting a molecular mechanism through which impaired insulin action and nuclear FOXO1 in IECs promotes CAC.
Subject(s)
Colonic Neoplasms/prevention & control , Forkhead Box Protein O1/metabolism , Insulin-Like Growth Factor I/metabolism , Insulin/metabolism , Intestinal Mucosa/metabolism , Animals , Colonic Neoplasms/metabolism , Diet, High-Fat , Gene Expression Regulation/physiology , Humans , Insulin/physiology , Intestinal Mucosa/cytology , Mice , Mice, Inbred C57BL , Signal TransductionABSTRACT
OBJECTIVE: Obesity represents a major risk factor for the development of type 2 diabetes mellitus, atherosclerosis and certain cancer entities. Treatment of obesity is hindered by the long-term maintenance of initially reduced body weight, and it remains unclear whether all pathologies associated with obesity are fully reversible even upon successfully maintained weight loss. METHODS: We compared high fat diet-fed, weight reduced and lean mice in terms of body weight development, adipose tissue and liver insulin sensitivity as well as inflammatory gene expression. Moreover, we assessed similar parameters in a human cohort before and after bariatric surgery. RESULTS: Compared to lean animals, mice that demonstrated successful weight reduction showed increased weight gain following exposure to ad libitum control diet. However, pair-feeding weight-reduced mice with lean controls efficiently stabilized body weight, indicating that hyperphagia was the predominant cause for the observed weight regain. Additionally, whereas glucose tolerance improved rapidly after weight loss, systemic insulin resistance was retained and ameliorated only upon prolonged pair-feeding. Weight loss enhanced insulin action and resolved pro-inflammatory gene expression exclusively in the liver, whereas visceral adipose tissue displayed no significant improvement of metabolic and inflammatory parameters compared to obese mice. Similarly, bariatric surgery in humans (n = 55) resulted in massive weight reduction, improved hepatic inflammation and systemic glucose homeostasis, while adipose tissue inflammation remained unaffected and adipocyte-autonomous insulin action only exhibit minor improvements in a subgroup of patients (42%). CONCLUSIONS: These results demonstrate that although sustained weight loss improves systemic glucose homeostasis, primarily through improved inflammation and insulin action in liver, a remarkable obesogenic memory can confer long-term increases in adipose tissue inflammation and insulin resistance in mice as well as in a significant subpopulation of obese patients.
Subject(s)
Brain/physiology , Energy Metabolism/physiology , Glucose Intolerance/physiopathology , Glucose/metabolism , Obesity/physiopathology , Animals , Diabetes Mellitus, Type 2/etiology , Diabetes Mellitus, Type 2/physiopathology , Diabetes Mellitus, Type 2/therapy , Feedback, Physiological/physiology , Ghrelin/physiology , Glucose Intolerance/etiology , Glucose Intolerance/therapy , Humans , Hypothalamus/physiology , Insulin/physiology , Leptin/physiology , Neurotransmitter Agents/physiology , Nutritional Status/physiology , Obesity/etiology , Obesity/therapyABSTRACT
Mutations in single genes and environmental interventions can extend healthy lifespan in laboratory model organisms. Some of the mechanisms involved show evolutionary conservation, opening the way to using simpler invertebrates to understand human ageing. Forkhead transcription factors have been found to play a key role in lifespan extension by alterations in the insulin/IGF pathway and by dietary restriction. Interventions that extend lifespan have also been found to delay or ameliorate the impact of ageing-related pathology and disease, including cancer. Understanding the mode of action of forkheads in this context will illuminate the mechanisms by which ageing acts as a risk factor for ageing-related disease, and could lead to the development of a broad-spectrum, preventative medicine for the diseases of ageing.
Subject(s)
Aging/physiology , Forkhead Transcription Factors/physiology , Animals , HumansABSTRACT
Insulin resistance and type 2 diabetes are serious public health threats. Although enormous research efforts have been focused on the pathogenesis of these diseases, the underlying mechanisms remain only partly understood. Here we review mouse phenotypes resulting from inactivation of molecules responsible for the control of glucose metabolism that have led to novel insights into insulin action and the development of insulin resistance. In addition, more sophisticated strategies to manipulate genes in mice in the future are presented.
Subject(s)
Diabetes Mellitus, Type 2/physiopathology , Disease Models, Animal , Insulin Resistance/physiology , Mice, Knockout , Mice, Transgenic , Animals , MiceABSTRACT
To understand the mechanism of insulin signalling and insulin resistance in the development of type 2 diabetes, it is necessary to elucidate the role of insulin and related signal molecules in normal cellular development and functions. A technique for addressing this problem, which is growing more and more important, is the generation and characterization of knockout animal models; such models allow in vivo study of the effects of a lack of a certain gene product, for example, a hormone or intracellular signalling molecule, on the viability, development and physiology of the animal. Besides the conventional form of knockout-which abolishes expression of the gene of interest in every cell of the body and during embryonic development-more recent technology permits the selective inactivation of genes in a tissue-specific and even time-controlled manner. With these techniques, it has become possible not only to examine the function of genes whose conventional inactivation would be lethal for the animal, but also to examine the specific functions that these genes have in certain tissues or at certain developmental stages. Here, we review the phenotype of mice resulting from both conventional and conditional inactivation of molecules in the insulin signalling cascade; this work has led to novel concepts in the understanding of insulin action and the development of insulin resistance.
Subject(s)
Diabetes Mellitus, Type 2/physiopathology , Insulin Resistance/genetics , Insulin/metabolism , Signal Transduction/physiology , Animals , Mice , Mice, KnockoutABSTRACT
Insulin receptors (IRs) and insulin signaling proteins are widely distributed throughout the central nervous system (CNS). To study the physiological role of insulin signaling in the brain, we created mice with a neuron-specific disruption of the IR gene (NIRKO mice). Inactivation of the IR had no impact on brain development or neuronal survival. However, female NIRKO mice showed increased food intake, and both male and female mice developed diet-sensitive obesity with increases in body fat and plasma leptin levels, mild insulin resistance, elevated plasma insulin levels, and hypertriglyceridemia. NIRKO mice also exhibited impaired spermatogenesis and ovarian follicle maturation because of hypothalamic dysregulation of luteinizing hormone. Thus, IR signaling in the CNS plays an important role in regulation of energy disposal, fuel metabolism, and reproduction.
Subject(s)
Body Weight , Brain/metabolism , Insulin/physiology , Receptor, Insulin/physiology , Reproduction , Adipose Tissue , Animals , Blood Glucose/analysis , Eating , Female , Hypertriglyceridemia/etiology , Insulin/blood , Insulin Resistance , Leptin/blood , Leuprolide/pharmacology , Luteinizing Hormone/blood , Male , Mice , Mice, Knockout , Neurons/metabolism , Obesity/etiology , Ovarian Follicle/physiology , Receptor, Insulin/genetics , Sex Characteristics , Signal Transduction , SpermatogenesisABSTRACT
The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70-80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration of egr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation.
Subject(s)
Insulin-Like Growth Factor I/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Phosphoproteins/physiology , 3T3 Cells , Adaptor Proteins, Signal Transducing , Animals , Cell Division , Enzyme Activation , Epidermal Growth Factor/pharmacology , Female , Genetic Vectors , Insulin Receptor Substrate Proteins , Mice , Mice, Knockout , Phosphoproteins/deficiency , Phosphoproteins/genetics , Phosphotyrosine/analysis , Recombinant Proteins/metabolism , Retroviridae , Signal Transduction/drug effects , Signal Transduction/physiology , TransfectionABSTRACT
Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.
Subject(s)
Genes, fos/genetics , Growth Substances/biosynthesis , Protein Serine-Threonine Kinases , Ribosomal Protein S6 Kinases/physiology , Transcription Factors , Transcription, Genetic , 3T3 Cells , Animals , DNA-Binding Proteins/metabolism , Fibroblasts/metabolism , Genes, Immediate-Early/genetics , Insulin-Like Growth Factor I/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/metabolism , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Serum Response Factor , Time Factors , Transfection , ets-Domain Protein Elk-1ABSTRACT
Induction of cfos expression is a definite end point of signal transduction by receptor tyrosine kinases via MAPK cascades. We have examined signal transduction to transcription factor cFos in isolated fibroblasts of a patient with an inherited syndrome of insulin resistance. MAPK phosphorylation and activity were unaltered, but inducibility of cfos transcription was strongly impaired by insulin and reduced by PDGF. Induction of the cfos promoter via MAPK is mediated by activation of the ternary complex. Abundance of SRF or Elk-1 was unaltered, but Elk-1 phosphorylation following stimulation was reduced. Transient transfections with reporter genes under control of the Elk-1 binding ets/sre cis element or expression plasmids coding for the regulatory domain of Elk-1 fused to heterologous DNA binding domains revealed a defect of Elk-1 activation in the patient cells. These data identify a novel postreceptor defect of insulin and growth factors involving activation of transcription.
Subject(s)
DNA-Binding Proteins , Insulin Resistance , Proto-Oncogene Proteins c-fos/genetics , Signal Transduction , Transcription Factors , Cells, Cultured , Fibroblasts/metabolism , Gene Expression Regulation , Humans , Mitogen-Activated Protein Kinases/metabolism , Protein Conformation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-fos/metabolism , Transcriptional Activation , ets-Domain Protein Elk-1ABSTRACT
Type 2 diabetes is characterized by abnormalities of insulin action in muscle, adipose tissue, and liver and by altered beta-cell function. To analyze the role of the insulin signaling pathway in these processes, we have generated mice with combined heterozygous null mutations in insulin receptor (ir), insulin receptor substrate (irs-1), and/or irs-2. Diabetes developed in 40% of ir/irs-1/irs-2(+/-), 20% of ir/irs-1(+/-), 17% of ir/irs-2(+/-), and 5% of ir(+/-) mice. Although combined heterozygosity for ir/irs-1(+/-) and ir/irs-2(+/-) results in a similar number of diabetic mice, there are significant differences in the underlying metabolic abnormalities. ir/irs-1(+/-) mice develop severe insulin resistance in skeletal muscle and liver, with compensatory beta-cell hyperplasia. In contrast, ir/irs-2(+/-) mice develop severe insulin resistance in liver, mild insulin resistance in skeletal muscle, and modest beta-cell hyperplasia. Triple heterozygotes develop severe insulin resistance in skeletal muscle and liver and marked beta-cell hyperplasia. These data indicate tissue-specific differences in the roles of IRSs to mediate insulin action, with irs-1 playing a prominent role in skeletal muscle and irs-2 in liver. They also provide a practical demonstration of the polygenic and genetically heterogeneous interactions underlying the inheritance of type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Insulin Resistance/genetics , Phosphoproteins/genetics , Receptor, Insulin/genetics , Adipose Tissue/enzymology , Animals , Blood Glucose/metabolism , Cell Size/genetics , Diabetes Mellitus, Type 2/blood , Disease Models, Animal , Heterozygote , Homozygote , Hyperglycemia/diagnosis , Hyperglycemia/genetics , Insulin/blood , Insulin Receptor Substrate Proteins , Intracellular Signaling Peptides and Proteins , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Liver/enzymology , Male , Mice , Mice, Knockout , Muscle, Skeletal/enzymology , Mutation , Organ Specificity/genetics , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
The failure of insulin to stimulate muscle glucose uptake and suppress hepatic glucose production represents two of the fundamental pathophysiologic lesions in type 2 diabetes mellitus (DM). Defining insulin action at the molecular level, therefore, provides the critical background against which to elucidate the mechanisms of insulin resistance that underlie type 2 DM, obesity and many other disorders. Over the past two decades substantial progress has been made in identifying many of the molecular mechanisms involved in insulin signaling. Much of this progress has been due to the use of homologous recombinant gene targeting. The present review examines the various insights that have been provided by studies of knockout mice strains. Taken together, the results present the possibility of a unifying hypothesis for type 2 DM, in which insulin resistance in the beta-cell synergizes with insulin resistance in the periphery to produce the two classic defects of this disease: relative hypoinsulinemia and peripheral insulin resistance.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus/genetics , Homeostasis , Mice, Knockout , Animals , Humans , Insulin/physiology , Insulin Receptor Substrate Proteins , Islets of Langerhans/physiology , Liver/physiology , Mice , Phosphoproteins/deficiency , Phosphoproteins/genetics , Phosphoproteins/physiology , Receptor, Insulin/deficiency , Receptor, Insulin/genetics , Receptor, Insulin/physiology , Signal TransductionABSTRACT
Insulin receptor substrate-1 (IRS-1) is pivotal in mediating the actions of insulin and growth factors in most tissues of the body, but its role in insulin-producing beta islet cells is unclear. Freshly isolated islets from IRS-1 knockout mice and SV40-transformed IRS-1-deficient beta-cell lines exhibit marked insulin secretory defects in response to glucose and arginine. Furthermore, insulin expression is reduced by about 2-fold in the IRS-1-null islets and beta-cell lines, and this defect can be partially restored by transfecting the cells with IRS-1. These data provide evidence for an important role of IRS-1 in islet function and provide a novel functional link between the insulin signaling and insulin secretion pathways. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.
Subject(s)
Islets of Langerhans/physiology , Phosphoproteins/physiology , Animals , Cell Line , Glucagon/metabolism , Insulin/analysis , Insulin/metabolism , Insulin Receptor Substrate Proteins , Insulin Secretion , Islets of Langerhans/chemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphoproteins/analysis , Phosphoproteins/deficiency , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
KNOCKOUT MICE: The generation of knockout mice has largely improved our understanding of the function of a variety of gene products. Gene inactivation experiments in mice have yielded numerous animal models for human diseases, thereby expanding our understanding of the underlying pathophysiological mechanisms. The use of conventional knockout experiments is limited if the phenotyp of gene disruption results in embryonic letality. CONDITIONAL MUTAGENESIS: Conditional mutagenesis aims to overcome this limitation by regional and temporal control of gene inactivation in mice. CRE-LOXP SYSTEM: The bacteriophage-enzyme Cre recognizes loxP-sites in the genome and excises loxP-flanked DNA-regions. Using this system loxP-sites can be introduced into intron regions of a target gene and mice can be created carrying this functional, but loxP-marked gene. When crossed with transgenic mice expressing the Cre-recombinase under control of a tissue-specific and/or inducible promoter the gene will be inactivated in vivo in a timely and regionally controlled fashion.
Subject(s)
Diabetes Mellitus, Experimental , Disease Models, Animal , Animals , Animals, Genetically Modified , Diabetes Mellitus, Experimental/genetics , Gene Targeting , Genetic Engineering , Humans , Mice , Mice, Knockout , Recombination, GeneticABSTRACT
Dysfunction of the pancreatic beta cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the beta cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the beta cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic beta cell and suggest that defects in insulin signaling at the level of the beta cell may contribute to the observed alterations in insulin secretion in type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Insulin/deficiency , Insulin/metabolism , Islets of Langerhans/metabolism , Receptor, Insulin/deficiency , Receptor, Insulin/genetics , Viral Proteins , Alleles , Animals , Blood Glucose/metabolism , Humans , Immunohistochemistry , Insulin/genetics , Insulin Secretion , Integrases/genetics , Islets of Langerhans/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Organ Specificity/genetics , Pancreas/metabolism , Pancreas/pathology , Pancreas/ultrastructure , Rats , TransgenesABSTRACT
Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.
Subject(s)
ErbB Receptors/metabolism , Phosphoproteins/metabolism , Tyrosine/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Base Sequence , Cell Fractionation , Humans , Kinetics , Molecular Sequence Data , Phosphoamino Acids/analysis , Phosphoproteins/genetics , Phosphorylation , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/biosynthesis , Tumor Cells, CulturedABSTRACT
Mutations of mitochondrial DNA (mtDNA) cause several well-recognized human genetic syndromes with deficient oxidative phosphorylation and may also have a role in ageing and acquired diseases of old age. We report here that hallmarks of mtDNA mutation disorders can be reproduced in the mouse using a conditional mutation strategy to manipulate the expression of the gene encoding mitochondrial transcription factor A (Tfam, previously named mtTFA), which regulates transcription and replication of mtDNA. Using a loxP-flanked Tfam allele (TfamloxP) in combination with a cre-recombinase transgene under control of the muscle creatinine kinase promoter, we have disrupted Tfam in heart and muscle. Mutant animals develop a mosaic cardiac-specific progressive respiratory chain deficiency, dilated cardiomyopathy, atrioventricular heart conduction blocks and die at 2-4 weeks of age. This animal model reproduces biochemical, morphological and physiological features of the dilated cardiomyopathy of Kearns-Sayre syndrome. Furthermore, our findings provide genetic evidence that the respiratory chain is critical for normal heart function.
Subject(s)
Cardiomyopathy, Dilated/genetics , DNA, Mitochondrial , DNA-Binding Proteins , Gene Expression Regulation , Heart Block/genetics , Heart/physiopathology , High Mobility Group Proteins , Mitochondrial Proteins , Nuclear Proteins , Trans-Activators , Transcription Factors/biosynthesis , Viral Proteins , Xenopus Proteins , Animals , Cardiomyopathy, Dilated/physiopathology , Creatine Kinase/genetics , Disease Models, Animal , Electron Transport Complex IV/metabolism , Female , Heart Block/physiopathology , Humans , Integrases/genetics , Male , Mice , Mice, Transgenic , Muscle, Skeletal , Myocardium , NAD(P)H Dehydrogenase (Quinone)/metabolism , Transcription Factors/geneticsABSTRACT
Skeletal muscle insulin resistance is among the earliest detectable defects in humans with type 2 diabetes mellitus. To determine the contribution of muscle insulin resistance to the metabolic phenotype of diabetes, we used the Cre-loxP system to disrupt the insulin receptor gene in mouse skeletal muscle. The muscle-specific insulin receptor knockout mice exhibit a muscle-specific > 95% reduction in receptor content and early signaling events. These mice display elevated fat mass, serum triglycerides, and free fatty acids, but blood glucose, serum insulin, and glucose tolerance are normal. Thus, insulin resistance in muscle contributes to the altered fat metabolism associated with type 2 diabetes, but tissues other than muscle appear to be more involved in insulin-regulated glucose disposal than previously recognized.