Thyroid Hormone Signaling In Vivo Requires a Balance between Coactivators and Corepressors

Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1^−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 (Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1^−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1^−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR^ΔID/ΔID Src-1^−/− mice have normal TH and TSH levels and are triiodothryonine (T₃) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T₃ activation of key hepatic gene targets, NCoR^ΔID/ΔID Src-1^−/− mice reacquired hepatic T₃ sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR^ΔID/ΔID Src-1^−/− mice, suggesting that SRC-2 is responsible for T₃ sensitivity in the absence of NCoR1 and SRC-1. Thus, T₃ targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1^−/− mice through increased SRC-2 recruitment to T₃ target genes.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Targeted Inactivation of p12Cdk2ap1, CDK2 Associating Protein 1, Leads to Early Embryonic Lethality

Targeted disruption of murine Cdk2ap1, an inhibitor of CDK2 function and hence G1/S transition, results in the embryonic lethality with a high penetration rate. Detailed timed pregnancy analysis of embryos showed that the lethality occurred between embryonic day 3.5 pc and 5.5 pc, a period of implantation and early development of implanted embryos. Two homozygous knockout mice that survived to term showed identical craniofacial defect, including a short snout and a round forehead. Examination of craniofacial morphology by measuring Snout Length (SL) vs. Face Width (FW) showed that the Cdk2ap1^+/− mice were born with a reduced SL/FW ratio compared to the Cdk2ap1^+/+ and the reduction was more pronounced in Cdk2ap1^−/− mice. A transgenic rescue of the lethality was attempted by crossing Cdk2ap1^+/− animals with K14-Cdk2ap1 transgenic mice. Resulting Cdk2ap1^{+/−:K14-Cdk2ap1} transgenic mice showed an improved incidence of full term animals (16.7% from 0.5%) on a Cdk2ap1^−/− background. Transgenic expression of Cdk2ap1 in Cdk2ap1^{−/−:K14-Cdk2ap1} animals restored SL/FW ratio to the level of Cdk2ap1^{+/−:K14-Cdk2ap1} mice, but not to that of the Cdk2ap1^{+/+:K14-Cdk2ap1} mice. Teratoma formation analysis using mESCs showed an abrogated in vivo pluripotency of Cdk2ap1^−/− mESCs towards a restricted mesoderm lineage specification. This study demonstrates that Cdk2ap1 plays an essential role in the early stage of embryogenesis and has a potential role during craniofacial morphogenesis.     

Thermodynamics of Formate-Oxidizing Metabolism and Implications for H2 Production

Formate-dependent proton reduction to H₂ (HCOO⁻ + H₂O → HCO₃⁻ + H₂) has been reported for hyperthermophilic Thermococcus strains. In this study, a hyperthermophilic archaeon, Thermococcus onnurineus strain NA1, yielded H₂ accumulation to a partial pressure of 1 × 10⁵ to 7 × 10⁵ Pa until the values of Gibbs free energy change (ΔG) reached near thermodynamic equilibrium (−1 to −3 kJ mol⁻¹). The bioenergetic requirement for the metabolism to conserve energy was demonstrated by ΔG values as small as −5 kJ mol⁻¹, which are less than the biological minimum energy quantum, −20 kJ mol⁻¹, as calculated by Schink (B. Schink, Microbiol. Mol. Biol. Rev. 61:262-280, 1997). Considering formate as a possible H₂ storage material, the H₂ production potential of the strain was assessed. The volumetric H₂ production rate increased linearly with increasing cell density, leading to 2,820 mmol liter⁻¹ h⁻¹ at an optical density at 600 nm (OD₆₀₀) of 18.6, and resulted in the high specific H₂ production rates of 404 ± 6 mmol g⁻¹ h⁻¹. The H₂ productivity indicates the great potential of T. onnurineus strain NA1 for practical application in comparison with H₂-producing microbes. Our result demonstrates that T. onnurineus strain NA1 has a highly efficient metabolic system to thrive on formate in hydrothermal systems.     

In Vivo 23Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

Sodium gradients (ΔpNa) were measured in resting cells of Fibrobacter succinogenes by in vivo ²³Na nuclear magnetic resonance using Tm(DOTP)⁵⁻ [thulium(III) 1,4,7,10-tetraazacyclododecane-N′,N′′,N′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ΔpNa of −55 to −40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na⁺ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).     

Novel Association of HK1 with Glycated Hemoglobin in a Non-Diabetic Population: A Genome-Wide Evaluation of 14,618 Participants in the Women's Genome Health Study

Type 2 diabetes is a leading cause of morbidity and mortality. While genetic variants have been found to influence the risk of type 2 diabetes mellitus, relatively few studies have focused on genes associated with glycated hemoglobin, an index of the mean blood glucose concentration of the preceding 8–12 weeks. Epidemiologic studies and randomized clinical trials have documented the relationship between glycated hemoglobin levels and the development of long-term complications in diabetes; moreover, higher glycated hemoglobin levels in the subdiabetic range have been shown to predict type 2 diabetes risk and cardiovascular disease. To examine the common genetic determinants of glycated hemoglobin levels, we performed a genome-wide association study that evaluated 337,343 SNPs in 14,618 apparently healthy Caucasian women. The results show that glycated hemoglobin levels are associated with genetic variation at the GCK (rs730497; P = 2.8×10⁻¹²), SLC30A8 (rs13266634; P = 9.8×10⁻⁸), G6PC2 (rs1402837; P = 6.8×10⁻¹⁰), and HK1 (rs7072268; P = 6.4×10⁻⁹) loci. While associations at the GCK, SLC30A8, and G6PC2 loci are confirmatory, the findings at HK1 are novel. We were able to replicate this novel association in an independent validation sample of 455 additional non-diabetic men and women. HK1 encodes the enzyme hexokinase, the first step in glycolysis and a likely candidate for the control of glucose metabolism. This observed genetic association between glycated hemoglobin levels and HK1 polymorphisms paves the way for further studies of the role of HK1 in hemoglobin glycation, glucose metabolism, and diabetes.     

Integrin α1-null Mice Exhibit Improved Fatty Liver When Fed a High Fat Diet Despite Severe Hepatic Insulin Resistance

Hepatic insulin resistance is associated with increased collagen. Integrin α1β1 is a collagen-binding receptor expressed on hepatocytes. Here, we show that expression of the α1 subunit is increased in hepatocytes isolated from high fat (HF)-fed mice. To determine whether the integrin α1 subunit protects against impairments in hepatic glucose metabolism, we analyzed glucose tolerance and insulin sensitivity in HF-fed integrin α1-null (itga1^−/−) and wild-type (itga1^+/+) littermates. Using the insulin clamp, we found that insulin-stimulated hepatic glucose production was suppressed by ∼50% in HF-fed itga1^+/+ mice. In contrast, it was not suppressed in HF-fed itga1^−/− mice, indicating severe hepatic insulin resistance. This was associated with decreased hepatic insulin signaling in HF-fed itga1^−/− mice. Interestingly, hepatic triglyceride and diglyceride contents were normalized to chow-fed levels in HF-fed itga1^−/− mice. This indicates that hepatic steatosis is dissociated from insulin resistance in HF-fed itga1^−/− mice. The decrease in hepatic lipid accumulation in HF-fed itga1^−/− mice was associated with altered free fatty acid metabolism. These studies establish a role for integrin signaling in facilitating hepatic insulin action while promoting lipid accumulation in mice challenged with a HF diet.     

Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO₂⁻) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO₂⁻-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO₂⁻ tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO₂⁻. Nitrite decreased the cellular NADH/NAD⁺ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO₂⁻-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO₂⁻. These findings demonstrate a link between NO₂⁻ tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO₂⁻-tolerating mechanism in this strain.     

ABCA1 in adipocytes regulates adipose tissue lipid content,glucose tolerance,and insulin sensitivity

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1^−ad/−ad). When fed a high-fat, high-cholesterol diet, ABCA1^−ad/−ad mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1^−ad/−ad mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.     

RHAMM Promotes Interphase Microtubule Instability and Mitotic Spindle Integrity through MEK1/ERK1/2 Activity

An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMM^Δ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMM^Δ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM^−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMM^Δ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMM^Δ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMM^Δ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMM^Δ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMM^Δ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMM^Δ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates.     

Comparison of Assimilatory Organic Nitrogen, Sulfur, and Carbon Sources for Growth of Methanobacterium Species

Experiments document the ability of two species of autotrophic methanogens to assimilate and utilize organic substrates as the nutrient sulfur or nitrogen source and as a carbon source during growth on H₂-CO₂. Methanobacterium thermoautotrophicum strain ΔH and the mesophilic species Methanobacterium sp. strain Ivanov grew with glutamine as the nitrogen source or cysteine as the sulfur source. M. thermoautotrophicum also utilized urea as the nitrogen source and as a carbon precursor for methane and cell synthesis. Methanobacterium sp. strain Ivanov grew with methionine as the sulfur source. The growth rate of two different Methanobacterium species was lower on an organic N or S source than on ammonium or sulfide. ³⁵S and ¹⁴C tracer studies demonstrated that amino acid or urea assimilation correlated with time and amount of growth. The rate of [³⁵S]cysteine incorporation was similar in strain ΔH (34 nmol h⁻¹ mg of cells⁻¹) and strain Ivanov (23 nmol h⁻¹ mg of cells⁻¹). However, the rate of [¹⁴C]acetate incorporation was dramatically different (17 versus 208 nmol h⁻¹ mg of cells⁻¹ in strains ΔH and Ivanov, respectively). [¹⁴C]acetate accounted for 1.3 and 21.2% of the total cell carbon synthesized by strains ΔH and Ivanov, respectively. Amino acids and urea were mainly assimilated into the cell protein fraction, but accounted for less than 2.0% of the total cell carbon synthesized. The data suggest that a biochemical-genetic approach to understanding cell carbon synthesis in methanogens is feasible; mutants that are auxotrophic for either acetate, glutamine, cysteine, or methionine are suggested as future targets for genetic studies.     

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α^−/−;Gsk-3β^−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α^−/−;Gsk-3β^−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.