Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo^−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.     

Nr5a1-Cre-mediated Tspo conditional knockout mice with low growth rate and prediabetes symptoms – A mouse model of stress diabetes

Translocator protein (TSPO) is a high-affinity cholesterol- and drug-binding mitochondrial protein. Nuclear receptor subfamily 5 group A member 1 or steroidogenic factor 1 (Nr5a1)-Cre mice were previously used to generate steroidogenic cell-specific Tspo gene conditional knockout (cKO) mice. TSPO-depleted homozygotes showed no response to adrenocorticotropic hormone (ACTH) in stimulating adrenal cortex corticosterone production but showed increased epinephrine synthesis in the medulla. No other phenotype was observed under normal growth conditions. During these studies, we noted that pairing two cKO mice resulted in the generation of small pups. These pups showed low growth rate at weaning, which has been linked to the development of type 2 diabetes (T2D) in adulthood. Experimental verification of T2D symptoms via blood testing of the adult mice, including glycated hemoglobin and insulin C-peptide measurements, showed that these Tspo cKO mice exhibited sustained hyperglycemia, a sign of prediabetes, likely due to the augmentation of hepatic glucose production mediated by the increased epinephrine. We also observed increased expression of the S100a8 gene, which is upregulated after chronic glucose stimulation. Taken together, the observed prediabetes phenotype and lack of response to ACTH indicate that Tspo cKO mice (Nr5a1-Cre^+/?, Tspo^fl/fl) could provide a useful model to study the link between diabetes and stress.     

Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.     

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.     

In Vivo Regulation of Steroid Hormones by the Chst10 Sulfotransferase in Mouse

Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10^−/− mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10^−/− females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10^−/− females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo.     

Lipocalin 2 Regulates Brown Fat Activation via a Nonadrenergic Activation Mechanism

In this study, we report that lipocalin 2 (Lcn2), a recently characterized adipokine/cytokine, is a novel regulator of brown adipose tissue (BAT) activation by modulating the adrenergic independent p38 MAPK-PGC-1α-UCP1 pathway. Global Lcn2 knock-out (Lcn2^−/−) mice have defective BAT thermogenic activation caused by cold stimulation and decreased BAT activity under high fat diet-induced obesity. Nevertheless, Lcn2^−/− mice maintain normal sympathetic nervous system activation as evidenced by normal catecholamine release and lipolytic activity in response to cold stimulation. Further studies showed that Lcn2 deficiency impairs peroxisomal and mitochondrial oxidation of lipids and attenuates cold-induced Pgc1a and Ucp1 expression and p38 MAPK phosphorylation in BAT. Moreover, in vitro studies showed that Lcn2 deficiency reduces the thermogenic activity of brown adipocytes. Lcn2^−/− differentiated brown adipocytes have significantly decreased expression levels of brown fat markers, decreased p38 MAPK phosphorylation, and decreased mitochondrial oxidation capacity. However, Lcn2^−/− brown adipocytes have normal norepinephrine-stimulated p38 MAPK and hormone-sensitive lipase phosphorylation and Pgc1a and Ucp1 expression, suggesting an intact β-adrenergic signaling activation. More intriguingly, recombinant Lcn2 was able to significantly stimulate p38 MAPK phosphorylation in brown adipocytes. Activating peroxisome proliferator-activated receptor γ, a downstream effector of PGC-1α, by thiazolidinedione administration fully reverses the BAT function of Lcn2^−/− mice. Our findings provide evidence for the novel role Lcn2 plays in oxidative metabolism and BAT activation via an adrenergic independent mechanism.     

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2^−/−) mice extended the life span of Tk2^−/− mice from 3 weeks to at least 20 months. The Dm-dNK^+/−Tk2^−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK^+/−Tk2^−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK^+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.     

Translocator Protein 2 Is Involved in Cholesterol Redistribution during Erythropoiesis

Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis.Translocator protein (TSPO)² is an 18-kDa protein that was previously known as PBR (peripheral type benzodiazepine receptor) and represents a gene family evolutionarily conserved from bacteria to humans (1). In bacteria, TSPO is the tryptophan-rich sensory protein, an integral membrane protein that acts as a negative regulator of the expression of specific photosynthesis genes in response to oxygen and light (2). It is involved in the efflux of porphyrin intermediates from the cell, and several conserved aromatic residues within TSPO are thought to be involved in binding porphyrin intermediates (2). TSPO of bacterial origin has been shown to have the same ligand binding properties as mammalian TSPO proteins (3). In addition to the binding of porphyrin and heme, mammalian TSPO can replace the activity of its bacterial homologs (2, 4, 5). Rat TSPO was shown to retain its structure within the bacterial outer membrane, to functionally substitute for the bacterial homolog, and to act in a manner similar to TSPO in the outer mitochondrial membrane (6). Therefore, it is conceivable that some conserved functions of the Tspo genes within a cell are maintained from bacteria to plants and to mammals.In mammals, the biological significance of TSPO has been studied for decades, and TSPO has been shown to be involved in a variety of cellular functions, including cholesterol transport and steroid hormone synthesis, mitochondrial respiration, permeability transition pore opening, apoptosis, and proliferation (7–10). Moreover, its expression correlates with certain pathological conditions such as cancer and endocrine and neurological diseases (8). Although some conserved cellular functions of Tspo are shared from bacteria to mammals, such as cholesterol-binding and transport, their biological significance seems to have adapted to serve specific functions critical for each organism. For instance, cholesterol transport into mitochondria is the rate-determining step in steroidogenesis (8, 11). TSPO serves the similar function in plants (12), insects (13), and mammals (14). However, the appearance of the drug, such as the benzodiazepine diazepam, binding sites on TSPO evolved later than the brain-specific γ-aminobutyric acid A receptor benzodiazepine binding sites (15), although drug binding was observed in both the plant and insect TSPOs (12, 13). Thus, throughout evolution, mammalian Tspo genes have exhibited extraordinary plasticity, a valuable trait to be further exploited.We sought to reveal the mechanisms controlling the molecular evolution of Tspo and Tspo-like genes and the ligand binding sites in recently sequenced mammalian and other eukaryotic genomes and characterize the relationships and potential functional similarities in cholesterol synthesis, trafficking, and cholesterol-supported steroidogenesis between different Tspo genes. During these studies, we identified a new family of Tspo-like genes involved in cholesterol trafficking and redistribution, which is linked to erythropoiesis and probably to a new mechanism of erythroblast maturation.     

Abcb10 Role in Heme Biosynthesis In Vivo: Abcb10 Knockout in Mice Causes Anemia with Protoporphyrin IX and Iron Accumulation

Abcb10, member 10 of the ABC transporter family, is reportedly a part of a complex in the mitochondrial inner membrane with mitoferrin-1 (Slc25a37) and ferrochelatase (Fech) and is responsible for heme biosynthesis in utero. However, it is unclear whether loss of Abcb10 causes pathological changes in adult mice. Here, we show that Abcb10^−/− mice lack heme biosynthesis and erythropoiesis abilities and die in midgestation. Moreover, we generated Abcb10^F/−; Mx1-Cre mice, with Abcb10 in hematopoietic cells deleted, which showed accumulation of protoporphyrin IX and maturation arrest in reticulocytes. Electron microscopy images of Abcb10^−/− hematopoietic cells showed a marked increase of iron deposits at the mitochondria. These results suggest a critical role for Abcb10 in heme biosynthesis and provide new insights into the pathogenesis of erythropoietic protoporphyria and sideroblastic anemia.     

Neurokinin-1 Receptor Signalling Impacts Bone Marrow Repopulation Efficiency

Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1^−/−) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 ^−/−, Tac4 ^−/− and Tac1 ^−/−/Tac4 ^−/− mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.     

p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47^phox knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47^phox−/− mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47^phox−/− mice but decreased in 2-year-old p47^phox−/− mice. Despite decreases in ROS generation in bone marrow cells and p47^phox-Nox2 signaling in osteoblastic cells, 2-year-old p47^phox−/− mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47^phox−/− mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47^phox-deficient mice occurs through an increased inflammatory milieu in bone and that p47^phox-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.     

Evaluation of Antiviral Efficacy of Ribavirin,Arbidol, and T-705 (Favipiravir) in a Mouse Model for Crimean-Congo Hemorrhagic Fever

Background

Mice lacking the type I interferon receptor (IFNAR^−/− mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR^−/− mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus.

Methodology/Principal Findings

CCHF virus-infected IFNAR^−/− mice died 2–6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC₅₀ 0.6–2.8 µg/ml; IC₉₀ 1.2–4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR^−/− mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects.

Conclusions/Significance

Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.     

Retinal Pigmented Epithelial Cells Obtained from Human Induced Pluripotent Stem Cells Possess Functional Visual Cycle Enzymes in Vitro and in Vivo

Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat^−/− and Rpe65^−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat^−/− and Rpe65^−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases.     

Defective Mitochondrial Function In Vivo in Skeletal Muscle in Adults with Down’s Syndrome: A 31P-MRS Study

Down’s syndrome (DS) is a developmental disorder associated with intellectual disability (ID). We have previously shown that people with DS engage in very low levels of exercise compared to people with ID not due to DS. Many aspects of the DS phenotype, such as dementia, low activity levels and poor muscle tone, are shared with disorders of mitochondrial origin, and mitochondrial dysfunction has been demonstrated in cultured DS tissue. We undertook a phosphorus magnetic resonance spectroscopy (³¹P-MRS) study in the quadriceps muscle of 14 people with DS and 11 non-DS ID controls to investigate the post-exercise resynthesis kinetics of phosphocreatine (PCr), which relies on mitochondrial respiratory function and yields a measure of muscle mitochondrial function in vivo. We found that the PCr recovery rate constant was significantly decreased in adults with DS compared to non-DS ID controls (1.7±0.1 min⁻¹ vs 2.1±0.1 min⁻¹ respectively) who were matched for physical activity levels, indicating that muscle mitochondrial function in vivo is impaired in DS. This is the first study to investigate mitochondrial function in vivo in DS using ³¹P-MRS. Our study is consistent with previous in vitro studies, supporting a theory of a global mitochondrial defect in DS.     

Cardiomyocyte-specific Loss of Diacylglycerol Acyltransferase 1 (DGAT1) Reproduces the Abnormalities in Lipids Found in Severe Heart Failure

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1^−/− mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1^−/−) mice. hDgat1^−/− mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1^−/− hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1^−/− mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1^−/−). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1^−/− mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1^−/− hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.