Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Early Pro-Inflammatory Remodeling of HDL Proteome in a Model of Diet-Induced Obesity: 2H2O-Metabolic Labeling-Based Kinetic Approach.

Mice fed a high-fat diet for 12 weeks or longer develop hyperglycemia, insulin resistance, dyslipidemia, and fatty liver. Additionally, a high-fat diet induces inflammation that remodels and affects the anti-inflammatory and antiatherogenic property of the high-density lipoprotein (HDL). However, the precise time course of metabolic disease progression and HDL remodeling remains unclear. Short-term (four weeks) high-fat feeding (60% fat calories) was performed in wild-type male C57BL/6J mice to gain insights into the early metabolic disease processes in conjunction with a HDL proteome dynamics analysis using a heavy water metabolic labeling approach. The high-fat diet-fed mice developed hyperglycemia, impaired glucose tolerance, hypercholesterolemia without hypertriglyceridemia or hepatic steatosis. A plasma HDL proteome dynamics analysis revealed increased turnover rates (and reduced half-lives) of several acute-phase response proteins involved in innate immunity, including complement C3 (12.77 ± 0.81 vs. 9.98 ± 1.20 h, p < 0.005), complement factor B (12.71 ± 1.01 vs. 10.85 ± 1.04 h, p < 0.05), complement Factor H (19.60 ± 1.84 vs. 16.80 ± 1.58 h, p < 0.05), and complement factor I (25.25 ± 1.29 vs. 19.88 ± 1.50 h, p < 0.005). Our findings suggest that an early immune response-induced inflammatory remodeling of the plasma HDL proteome precedes the diet-induced steatosis and dyslipidemia.

Utilization of High-Intensity Statins in Patients at Risk for Cardiovascular Events: A National Cross-Sectional Study.

BACKGROUND: The 2013 American College of Cardiology/American Heart Association cholesterol guidelines, which identified four groups of patients at risk for atherosclerotic cardiovascular disease events, departed from the target-based approach to managing cholesterol. The impact of these guidelines on high-intensity statin use across the United States is unclear. STUDY QUESTION: The primary objective was to evaluate the rate of high-intensity potential (HIP) statin use before and after the 2013 guidelines. The secondary objective was to identify predictors of HIP statin use within the study population. STUDY DESIGN: A national cross-sectional study was conducted using data from the National Ambulatory Medical Care Survey. Office visits involving patients aged 21-75 years where criteria for HIP statin therapy were met were included. Visits involving pregnant patients were excluded. MEASURES AND OUTCOMES: Prescribing trends of HIP statins were measured from National Ambulatory Medical Care Survey data before and after the 2013 guidelines. Multivariate logistic regression identified variables associated with prescribing HIP statins. RESULTS: A total of 48,884 visits were included, representing more than 940 million office visits nationally. HIP statins were listed in 9.5% and 16.5% of visits before and after 2013, respectively (odds ratio [OR] 1.88; 95% confidence interval [CI] 1.62-2.20). The strongest predictors of HIP statin use were antihypertensive use (OR 5.38, 95% CI 4.67-6.20), comorbid hyperlipidemia (OR 2.93, 95% CI 2.62-3.29), Black race (OR 0.63, 95% CI 0.49-0.81), and Hispanic ethnicity (OR 0.65, 95% CI 0.52-0.80). CONCLUSIONS: Prescribing rates for HIP statins increased after the release of the 2013 guidelines. The prescribing rates were lower than expected, especially in Black and Hispanic patients. These observations signify opportunities to improve the quality of care for patients who are at risk for atherosclerotic cardiovascular disease events in the United States.

Calculation of the Protein Turnover Rate Using the Number of Incorporated 2H Atoms and Proteomics Analysis of a Single Labeled Sample.

Rate constant estimation with heavy water requires a long-term experiment with data collection at multiple time points (3-4 weeks for mitochondrial proteome dynamics in mice and much longer in other species). When tissue proteins are analyzed, this approach requires euthanizing animals at each time point or multiple tissue biopsies in humans. Although short-term protocols are available, they require knowledge of the maximum number of isotope labels (N) and accurate quantification of observed 2H-enrichment in the peptide. The high-resolution accurate mass spectrometers used for proteome dynamics studies are characterized by a systematic spectral error that compromises these measurements. To circumvent these issues, we developed a simple algorithm for the rate constant calculation based on a single labeled sample and comparable unlabeled (time 0) sample. The algorithm determines N for all proteogenic amino acids from a long-term experiment to calculate the predicted plateau 2H-labeling of peptides for a short-term protocol and estimates the rate constant based on the measured baseline and the predicted plateau 2H-labeling of peptides. The method was validated based on the rate constant estimation in a long-term experiment in mice and dogs. The improved 2 time-point method enables the rate constant calculation with less than 10% relative error compared to the bench-marked multi-point method in mice and dogs and allows us to detect diet-induced subtle changes in ApoAI turnover in mice. In conclusion, we have developed and validated a new algorithm for protein rate constant calculation based on 2-time point measurements that could also be applied to other biomolecules.

AMPK-SIRT1-independent inhibition of ANGPTL3 gene expression is a potential lipid-lowering mechanism of metformin.

OBJECTIVES: Hypertriglyceridaemia enhances cardiovascular disease risk in patients with diabetes. Lipoprotein lipase (LPL) regulates plasma triglyceride levels by hydrolysing chylomicrons and very-low-density lipoprotein (VLDL). Metformin, an antidiabetic drug, improves plasma lipids including triglycerides. We examined metformin's regulation of angiopoietin-like 3 (ANGPTL3), a liver-derived secretory protein with LPL inhibitory property. METHODS: Using HepG2 cells, a human hepatocyte cell line, the effects of metformin on ANGPTL3 gene and protein expression were determined. The role of AMPK-SIRT1 pathway in metformin regulation of ANGPTL3 was determined using pharmacological, RNAi and reporter assays. Metformin regulation of ANGPTL3 expression was also examined in sodium palmitate-induced insulin resistance. KEY FINDINGS: Metformin and pharmacological activators of AMPK and SIRT1 inhibited the expression of ANGPTL3 in HepG2 cells. Pharmacological or RNAi-based antagonism of AMPK or SIRT1 failed to affect metformin inhibition of ANGPTL3. AMPK-SIRT1 activators and metformin exhibited distinct effects on the expression of ANGPTL3 gene luciferase reporter. Sodium palmitate-induced insulin resistance in cells resulted in increased ANGPTL3 gene expression which was suppressed by pretreatment with metformin. CONCLUSIONS: Metformin inhibits ANGPTL3 expression in the liver in an AMPK-SIRT1-independent manner as a potential mechanism to regulate LPL and lower plasma lipids.

Prevalence and Predictors of Benzodiazepine Monotherapy in Patients With Depression: A National Cross-Sectional Study.

OBJECTIVE: Depression guidelines discourage benzodiazepine monotherapy and limit use to short-term adjunctive therapy with antidepressants; however, patients with depression continue to receive benzodiazepine monotherapy. The prevalence and predictors of this prescribing pattern have not been described previously and are warranted to assist clinicians in identifying patients at highest risk of receiving benzodiazepine monotherapy. METHODS: A national, cross-sectional analysis of the National Ambulatory Medical Care Survey from 2012 to 2015 was performed for adults treated for depression. Depression was identified using a survey item specifically assessing the presence of depression. Office visits involving patients with bipolar disorder, schizoaffective disorder, or pregnancy were identified by ICD-9 code or specific survey item and were excluded. The primary endpoint was benzodiazepine monotherapy prescribing rate defined as initiation or continuation of a benzodiazepine in the absence of any antidepressant agent. A multivariate logistic regression model was created to identify variables associated with benzodiazepine monotherapy. RESULTS: In total, 9,426 unweighted visits were eligible for inclusion. Benzodiazepine monotherapy was identified in 9.3% of patients treated for depression (95% CI, 8.2%-10.6%). Predictors of benzodiazepine monotherapy included age of 45-64 years (OR = 1.39; 95% CI, 1.01-1.91), epilepsy-related office visit (OR = 5.34; 95% CI, 1.39-20.44), anxiety-related office visit (OR = 1.67; 95% CI, 1.23-2.27), underlying pulmonary disease (OR = 1.43; 95% CI, 1.09-1.87), and concomitant opiate prescribing (OR = 2.86; 95% CI, 2.01-4.06). Psychiatrists were less likely to prescribe benzodiazepine monotherapy than were other providers (OR = 0.42; 95% CI, 0.29-0.61). CONCLUSIONS: Benzodiazepine monotherapy is utilized in nearly 1 in 10 patients treated for depression. Adults aged 45 to 65 years, patients prescribed opioids, patients seen by primary care providers, and those with underlying anxiety, epilepsy, or pulmonary disorders are at highest risk.

Novel compounds that target lipoprotein lipase and mediate growth arrest in acute lymphoblastic leukemia.

Over the past decade, the therapeutic strategies employed to treat B-precursor acute lymphoblastic leukemia (ALL) have been progressively successful in treating the disease. Unfortunately, the treatment associated dyslipidemia, either acute or chronic, is very prevalent and a cause for decreased quality of life in the surviving patients. To overcome this hurdle, we tested a series of cylopropanecarboxamides, a family demonstrated to target lipid metabolism, for their anti-leukemic activity in ALL. Several of the compounds tested showed anti-proliferative activity, with one, compound 22, inhibiting both Philadelphia chromosome negative REH and Philadelphia chromosome positive SupB15 ALL cell division. The novel advantage of these compounds is the potential synergy with standard chemotherapeutic agents, while concomitantly blunting the emergence of dyslipidemia. Thus, the cylopropanecarboxamides represent a novel class of compounds that can be potentially used in combination with the present standard-of-care to limit treatment associated dyslipidemia in ALL patients.

Signal Transduction Mechanisms of Alcoholic Fatty Liver Disease: Emer ging Role of Lipin-1.

Lipin-1, a mammalian phosphatidic acid phosphatase (PAP), is a bi-functional molecule involved in various signaling pathways via its function as a PAP enzyme in the triglyceride synthesis pathway and in the nucleus as a transcriptional co-regulator. In the liver, lipin-1 is known to play a vital role in controlling the lipid metabolism and inflammation process at multiple regulatory levels. Alcoholic fatty liver disease (AFLD) is one of the earliest forms of liver injury and approximately 8-20% of patients with simple steatosis can develop into more severe forms of liver injury, including steatohepatitis, fibrosis/ cirrhosis, and eventually hepatocellular carcinoma (HCC). The signal transduction mechanisms for alcohol-induced detrimental effects in liver involves alteration of complex and multiple signaling pathways largely governed by a central and upstream signaling system, namely, sirtuin 1 (SIRT1)-AMP activated kinase (AMPK) axis. Emerging evidence suggests a pivotal role of lipin-1 as a crucial downstream regulator of SIRT1-AMPK signaling system that is likely to be ultimately responsible for development and progression of AFLD. Several lines of evidence demonstrate that ethanol exposure significantly induces lipin-1 gene and protein expression levels in cultured hepatocytes and in the livers of rodents, induces lipin-1-PAP activity, impairs the functional activity of nuclear lipin-1, disrupts lipin-1 mRNA alternative splicing and induces lipin-1 nucleocytoplasmic shuttling. Such impairment in response to ethanol leads to derangement of hepatic lipid metabolism, and excessive production of inflammatory cytokines in the livers of the rodents and human alcoholics. This review summarizes current knowledge about the role of lipin-1 in the pathogenesis of AFLD and its potential signal transduction mechanisms.

Emerging strategies of targeting lipoprotein lipase for metabolic and cardiovascular diseases.

Although statins and other pharmacological approaches have improved the management of lipid abnormalities, there exists a need for newer treatment modalities especially for the management of hypertriglyceridemia. Lipoprotein lipase (LPL), by promoting hydrolytic cleavage of the triglyceride core of lipoproteins, is a crucial node in the management of plasma lipid levels. Although LPL expression and activity modulation is observed as a pleiotropic action of some the commonly used lipid lowering drugs, the deliberate development of drugs targeting LPL has not occurred yet. In this review, we present the biology of LPL, highlight the LPL modulation property of currently used drugs and review the novel emerging approaches to target LPL.

Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator.

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.

Gelucire-stabilized nanoparticles as a potential DNA delivery system.

Clinical viability of gene delivery systems has been greatly impacted by potential toxicity of the delivery systems. Recently, we reported the nanoparticle (NP) preparation process that employs biocompatible materials such as Gelucire® 44/14 and cetyl alcohol as matrix materials. In the current study, the NP preparation was modified for pDNA loading through: (i) inclusion of cationic lipids (DOTAP or DDAB) with NP matrix materials; or (ii) application of cationic surfactants (CTAB) to generate NPs with desired surface charges for pDNA complexation. Colloidal stability and efficiency of loading pGL3-DR4X2-luciferase plasmid DNA in NPs were verified by gel permeation chromatography. Compared to pDNA alone, all the NPs were effective in preserving pDNA from digestion by DNase. While pDNA loading using CTAB-NPs involved fewer steps compared to DOTAP-NPs and DDAB-NPs, CTAB-NPs were greatly impacted by elevated cytotoxicity level which could be ascribed to the concentrations of CTAB in NP formulations. In vitro transfection studies (in HepG2 cells) based on luciferase expression showed the ranking of cell transfection efficiency as DOTAP-NPs > DDAB-NPs > CTAB-NPs. The overall work provided an initial assessment of gelucire-stabilized NPs as a potential platform for gene delivery.

Anti-edema action of thyroid hormone in MCAO model of ischemic brain stroke: Possible association with AQP4 modulation.

The use of neuroprotective strategies to mitigate the fatal consequences of ischemic brain stroke is a focus of robust research activity. We have previously demonstrated that thyroid hormone (T3; 3,3',5-triiodo-l-thyronine) possesses neuroprotective and anti-edema activity in pre-stroke treatment regimens when administered as a solution or as a nanoparticle formulation. In this study we have extended our evaluation of thyroid hormone use in animal models of brain stroke. We have used both transient middle cerebral artery occlusion (t-MCAO) and permanent (p-MCAO) models of ischemic brain stroke. A significant reduction of tissue infarction and a concurrent decrease in edema were observed in the t-MCAO model of brain stroke. However, no benefit of T3 was observed in p-MCAO stroke setting. Significant improvement of neurological outcomes was observed upon T3 treatment in t-MCAO mice. Further, we tested T2 (3,5-diiodo-l-thyronine) a natural deiodination metabolite of T3 in MCAO model of brain stroke. T2 potently decreased infarct size as well as edema formation. Additionally, we report here that T3 suppresses the expression of aquaporin-4 (AQP4) water channels which could be a likely mechanism of its anti-edema activity. Our studies provide evidence to stimulate clinical development of thyroid hormones for use in ischemic brain stroke.

A novel Lipoprotein lipase (LPL) agonist rescues the enzyme from inhibition by angiopoietin-like 4 (ANGPTL4).

Lipoprotein lipase (LPL) is a key physiological regulator of triglycerides and atherosclerosis risk. Random screening identified a compound designated C10, showing greater LPL agonist activity than NO-1886, a known LPL agonist. Structure-activity relationship (SAR) exploration of C10 led to the identification of C10d exhibiting at least two fold greater LPL activation than NO-1886. Unlike NO-1886, novel LPL agonists C10 and C10d reversed the LPL inhibition by angiopoietin-like 4 (ANGPTL4), a physiological inhibitor of LPL.

Engineering triiodothyronine (T3) nanoparticle for use in ischemic brain stroke.

A potential means of pharmacological management of ischemic stroke is rapid intervention using potent neuroprotective agents. Thyroid hormone (T3) has been shown to protect against ischemic damage in middle cerebral artery occlusion (MCAO) model of ischemic brain stroke. While thyroid hormone is permeable across the blood-brain barrier, we hypothesized that efficacy of thyroid hormone in ischemic brain stroke can be enhanced by encapsulation in nanoparticulate delivery vehicles. We tested our hypothesis by generating poly-(lactide-co-glycolide)-polyethyleneglycol (PLGA-b-PEG) nanoparticles that are either coated with glutathione or are not coated. We have previously reported that glutathione coating of PLGA-PEG nanoparticles is an efficient means of brain targeted drug delivery. Encapsulation of T3 in PLGA-PEG delivery vehicle resulted in particles that were in the nano range and exhibited a zeta potential of -6.51 mV (uncoated) or -1.70 mV (coated). We observed that both glutathione-coated and uncoated nanoparticles are taken up in cells wherein they stimulated the expression of thyroid hormone response element driven reporter robustly. In MCAO model of ischemic stroke, significant benefit of administering T3 in nanoparticulate form was observed over injection of a T3 solution. A 34 % decrease in tissue infarction and a 59 % decrease in brain edema were seen upon administration of T3 solution in MCAO stroke model. Corresponding measurements for uncoated T3 nanoparticles were 51 % and 68 %, whereas for the glutathione coated were 58 % and 75 %. Our study demonstrates that using nanoparticle formulations can significantly improve the efficacy of neuroprotective drugs in ischemic brain stroke.

Identification of a novel serum and glucocorticoid regulated kinase-1 (SGK1) ligand from virtual screening.

The serum and glucocorticoid regulated kinase-1 (SGK1) is part of the serine/threonine kinase family and has therapeutic potential in several neurodegenerative diseases such as ischemic stroke and Parkinson's disease. Here we use structure-based virtual screening to identify a novel ligand which inhibits SGK1 activity. The data presented here can be used for future scaffold hopping and possible drug development efforts.

Noncanonical mechanisms to regulate nuclear receptor signaling.

Nuclear receptor (NR)-targeted therapies comprise a large class of clinically employed drugs. A number of drugs currently being used against this protein class were designed as structural analogs of the endogenous ligand of these receptors. In recent years, there has been significant interest in developing newer strategies to target NRs, especially those that rely on mechanistic pathways of NR function. Prominent among these are noncanonical means of targeting NRs, which include selective NR modulation, NR coactivator interaction inhibition, inhibition of NR DNA binding, modulation of NR cellular localization, modulation of NR ligand biosynthesis and downregulation of NR levels in target tissues. This article reviews each of these promising emerging strategies for NR drug development and highlights some of most significant successes achieved in using them.

Similarities and differences between two modes of antagonism of the thyroid hormone receptor.

Thyroid hormone (T3) mediates diverse physiological functions including growth, differentiation, and energy homeostasis through the thyroid hormone receptors (TR). The TR binds DNA at specific recognition sequences in the promoter regions of their target genes known as the thyroid hormone response elements (TREs). Gene expression at TREs regulated by TRs is mediated by coregulator recruitment to the DNA bound receptor. This TR-coregulator interaction controls transcription of target genes by multiple mechanisms including covalent histone modifications and chromatin remodeling. Our previous studies identified a ß-aminoketone as a potent inhibitor of the TR-coactivator interaction. We describe here the activity of one of these inhibitors in modulating effects of T3 signaling in comparison to an established ligand-competitive inhibitor of TR, NH-3. The ß-aminoketone was found to reverse thyroid hormone induced gene expression by inhibiting coactivator recruitment at target gene promoters, thereby regulating downstream effects of thyroid hormone. While mimicking the downstream effects of NH-3 at the molecular level, the ß-aminoketone affects only a subset of the thyroid responsive signaling network. Thus antagonists directed to the coregulator binding site have distinct pharmacological properties relative to ligand-based antagonists and may provide complementary activity in vivo.

Characterization of the transactivation domain in the peroxisome-proliferator-activated receptor gamma co-activator (PGC-1).

The PGC-1s (peroxisome-proliferator-activated receptor gamma co-activators) are a family of transcriptional regulators that induce the expression of various metabolic genes. PGC-1 proteins stimulate genes involved in mitochondrial biogenesis, fatty acid oxidation and hepatic gluconeogenesis. Previous studies have demonstrated that the PGC-1alpha and beta isoforms interact with nuclear receptors through the conserved LXXLL (leucine-X-X-leucine-leucine) motifs. In the present study, we have investigated the mechanisms by which these PGC-1 isoforms stimulate gene expression. We have determined that the N-terminus of PGC-1 is responsible for transcriptional activation. Two conserved peptide motifs were identified in the N-terminus of PGC-1alpha and beta isoforms. These domains were named AD1 and AD2 (activation domain 1 and 2). Deletion of both of these motifs decreased the induction of various PGC-1-regulated genes including the PEPCK (phosphoenolpyruvate carboxykinase) and CPT-I (carnitine palmitoyltransferase-I) genes. It was determined that amino acids containing a negative charge in AD1 and the leucine residues in AD2 were important for the transcriptional induction of the PEPCK and CPT-I genes. Disruption of the AD motifs did not diminish the ability of the PGC-1alpha protein to associate with the PEPCK or CPT-I genes. In addition, deletion of the AD domains did not eliminate the ability of PGC-1alpha to interact with the thyroid hormone receptor. The data indicate that the AD1 and AD2 motifs mediate the induction of many PGC-1- responsive genes, but they do not contribute to the recruitment of PGC-1 to target genes.

Regulation of carnitine palmitoyltransferase I (CPT-Ialpha) gene expression by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) isoforms.

The peroxisome proliferator activated receptor gamma coactivators (PGC-1) have important roles in mitochondrial biogenesis and metabolic control in a variety of tissues. There are multiple isoforms of PGC-1 including PGC-1alpha and PGC-1beta. Both the PGC-1alpha and beta isoforms promote mitochondrial biogenesis and fatty acid oxidation, but only PGC-1alpha stimulates gluconeogenesis in the liver. Carnitine palmitoyltransferase I (CPT-I) is a key enzyme regulating mitochondrial fatty acid oxidation. In these studies, we determined that PGC-1beta stimulated expression of the "liver" isoform of CPT-I (CPT-Ialpha) but that PGC-1beta did not induce pyruvate dehydrogenase kinase 4 (PDK4) which is a regulator of pyruvate metabolism. The CPT-Ialpha gene is induced by thyroid hormone. We found that T3 increased the expression of PGC-1beta and that PGC-1beta enhanced the T3 induction of CPT-Ialpha. The thyroid hormone receptor interacts with PGC-1beta in a ligand dependent manner. Unlike PGC-1alpha, the interaction of PGC-1beta and the T3 receptor does not occur exclusively through the leucine-X-X-leucine-leucine motif in PGC-1beta. We have found that PGC-1beta is associated with the CPT-Ialpha gene in vivo. Overall, our results demonstrate that PGC-1beta is a coactivator in the T3 induction of CPT-Ialpha and that PGC-1beta has similarities and differences with the PGC-1alpha isoform.

Estrogen-related receptors stimulate pyruvate dehydrogenase kinase isoform 4 gene expression.

The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl-CoA in mitochondria and is a key regulatory enzyme in the oxidation of glucose to acetyl-CoA. Phosphorylation of PDC by the pyruvate dehydrogenase kinases (PDK2 and PDK4) inhibits PDC activity. Expression of the PDK genes is elevated in diabetes, leading to the decreased oxidation of pyruvate to acetyl-CoA. In these studies we have investigated the transcriptional regulation of the PDK4 gene by the estrogen-related receptors (ERRalpha and ERRgamma). The ERRs are orphan nuclear receptors whose physiological roles include the induction of fatty acid oxidation in heart and muscle. Previously, we found that the peroxisome proliferator-activated receptor gamma coactivator (PGC-1alpha) stimulates the expression of PDK4. Here we report that ERRalpha and ERRgamma stimulate the PDK4 gene in hepatoma cells, suggesting a novel role for ERRs in controlling pyruvate metabolism. In addition, both ERR isoforms recruit PGC-1alpha to the PDK4 promoter. Insulin, which decreases the expression of the PDK4 gene, inhibits the induction of PDK4 by ERRalpha and ERRgamma. The forkhead transcription factor (FoxO1) binds the PDK4 gene and contributes to the induction of PDK4 by ERRs and PGC-1alpha. Insulin suppresses PDK4 expression in part through the dissociation of FoxO1 and PGC-1alpha from the PDK4 promoter. Our data demonstrate a key role for the ERRs in the induction of hepatic PDK4 gene expression.