Liraglutide alleviates experimental diabetic cardiomyopathy in a PDH-dependent manner.

Liraglutide, a glucagon-like peptide-1 receptor (GLP-1R) agonist used for the treatment of T2D, has been shown to alleviate diabetic cardiomyopathy (DbCM) in experimental T2D, which was associated with increased myocardial glucose oxidation. To determine whether this increase in glucose oxidation is necessary for cardioprotection, we hypothesized that liraglutide's ability to alleviate DbCM would be abolished in mice with cardiomyocyte-specific deletion of pyruvate dehydrogenase (PDH; Pdha1CM-/- mice), the rate-limiting enzyme of glucose oxidation. Male Pdha1CM-/- mice and their α-myosin heavy chain Cre expressing littermates (αMHCCre mice) were subjected to experimental T2D via 10 weeks of high-fat diet supplementation, with a single low-dose injection of streptozotocin (75 mg/kg) provided at week 4. All mice were randomized to treatment with either vehicle control or liraglutide (30 µg/kg) twice daily during the final 2.5 weeks, with cardiac function assessed via ultrasound echocardiography. As expected, liraglutide treatment improved glucose homeostasis in both αMHCCre and Pdha1CM-/- mice with T2D, in the presence of mild weight loss. Parameters of systolic function were unaffected by liraglutide treatment in both αMHCCre and Pdha1CM-/- mice with T2D. However, liraglutide treatment alleviated diastolic dysfunction in αMHCCre mice, as indicated by an increase and decrease in the e'/a' and E/e' ratios, respectively. Conversely, liraglutide failed to rescue these indices of diastolic dysfunction in Pdha1CM-/- mice. Our findings suggest that increases in glucose oxidation are necessary for GLP-1R agonist mediated alleviation of DbCM. As such, strategies aimed at increasing PDH activity may represent a novel approach for the treatment of DbCM.

Loss of muscle PDH induces lactic acidosis and adaptive anaplerotic compensation via pyruvate-alanine cycling and glutaminolysis.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation that links glycolysis-derived pyruvate with the tricarboxylic acid (TCA) cycle. Although skeletal muscle is a significant site for glucose oxidation and is closely linked with metabolic flexibility, the importance of muscle PDH during rest and exercise has yet to be fully elucidated. Here, we demonstrate that mice with muscle-specific deletion of PDH exhibit rapid weight loss and suffer from severe lactic acidosis, ultimately leading to early mortality under low-fat diet provision. Furthermore, loss of muscle PDH induces adaptive anaplerotic compensation by increasing pyruvate-alanine cycling and glutaminolysis. Interestingly, high-fat diet supplementation effectively abolishes early mortality and rescues the overt metabolic phenotype induced by muscle PDH deficiency. Despite increased reliance on fatty acid oxidation during high-fat diet provision, loss of muscle PDH worsens exercise performance and induces lactic acidosis. These observations illustrate the importance of muscle PDH in maintaining metabolic flexibility and preventing the development of metabolic disorders.

Ketone ester administration improves glycemia in obese mice.

During periods of prolonged fasting/starvation, the liver generates ketones [i.e., ß-hydroxybutyrate (ßOHB)] that primarily serve as alternative substrates for ATP production. Previous studies have demonstrated that elevations in skeletal muscle ketone oxidation contribute to obesity-related hyperglycemia, whereas inhibition of succinyl CoA:3-ketoacid CoA transferase (SCOT), the rate-limiting enzyme of ketone oxidation, can alleviate obesity-related hyperglycemia. As circulating ketone levels are a key determinant of ketone oxidation rates, we tested the hypothesis that increases in circulating ketone levels would worsen glucose homeostasis secondary to increases in muscle ketone oxidation. Accordingly, male C57BL/6J mice were subjected to high-fat diet-induced obesity, whereas their lean counterparts received a standard chow diet. Lean and obese mice were orally administered either a ketone ester (KE) or placebo, followed by a glucose tolerance test. In tandem, we conducted isolated islet perifusion experiments to quantify insulin secretion in response to ketones. We observed that exogenous KE administration robustly increases circulating ßOHB levels, which was associated with an improvement in glucose tolerance only in obese mice. These observations were independent of muscle ketone oxidation, as they were replicated in mice with a skeletal muscle-specific SCOT deficiency. Furthermore, the R-isomer of ßOHB produced greater increases in perifusion insulin levels versus the S-isomer in isolated islets from obese mice. Taken together, acute elevations in circulating ketones promote glucose-lowering in obesity. Given that only the R-isomer of ßOHB is oxidized, further studies are warranted to delineate the precise role of ß-cell ketone oxidation in regulating insulin secretion.NEW & NOTEWORTHY It has been demonstrated that increased skeletal muscle ketone metabolism contributes to obesity-related hyperglycemia. Since increases in ketone supply are key determinants of organ ketone oxidation rates, we determined whether acute elevations in circulating ketones following administration of an oral ketone ester may worsen glucose homeostasis in lean or obese mice. Our work demonstrates the opposite, as acute elevations in circulating ketones improved glucose tolerance in obese mice.

The antianginal ranolazine fails to improve glycaemia in obese liver-specific pyruvate dehydrogenase deficient male mice.

AIMS: Recent studies have demonstrated that stimulating pyruvate dehydrogenase (PDH, gene Pdha1), the rate-limiting enzyme of glucose oxidation, can reverse obesity-induced non-alcoholic fatty liver disease (NAFLD), which can be achieved via treatment with the antianginal ranolazine. Accordingly, our aim was to determine whether ranolazine's ability to mitigate obesity-induced NAFLD and hyperglycaemia requires increases in hepatic PDH activity. METHODS: We generated liver-specific PDH-deficient (Pdha1Liver-/- ) mice, which were provided a high-fat diet for 12 weeks to induce obesity. Pdha1Liver-/- mice and their albumin-Cre (AlbCre ) littermates were randomized to treatment with either vehicle control or ranolazine (50 mg/kg) once daily via oral gavage during the final 5 weeks, following which we assessed glucose and pyruvate tolerance. RESULTS: Pdha1Liver-/- mice exhibited no overt phenotypic differences (e.g. adiposity, glucose tolerance) when compared to their AlbCre littermates. Of interest, ranolazine treatment improved glucose tolerance and mildly reduced hepatic triacylglycerol content in obese AlbCre mice but not in obese Pdha1Liver-/- mice. The latter was independent of changes in hepatic mRNA expression of genes involved in regulating lipogenesis. CONCLUSIONS: Liver-specific PDH deficiency is insufficient to promote an NAFLD phenotype. Nonetheless, hepatic PDH activity partially contributes to how the antianginal ranolazine improves glucose tolerance and alleviates hepatic steatosis in obesity.

The Antipsychotic Dopamine 2 Receptor Antagonist Diphenylbutylpiperidines Improve Glycemia in Experimental Obesity by Inhibiting Succinyl-CoA:3-Ketoacid CoA Transferase.

Despite significant progress in understanding the pathogenesis of type 2 diabetes (T2D), the condition remains difficult to manage. Hence, new therapeutic options targeting unique mechanisms of action are required. We have previously observed that elevated skeletal muscle succinyl CoA:3-ketoacid CoA transferase (SCOT) activity, the rate-limiting enzyme of ketone oxidation, contributes to the hyperglycemia characterizing obesity and T2D. Moreover, we identified that the typical antipsychotic agent pimozide is a SCOT inhibitor that can alleviate obesity-induced hyperglycemia. We now extend those observations here, using computer-assisted in silico modeling and in vivo pharmacology studies that highlight SCOT as a noncanonical target shared among the diphenylbutylpiperidine (DPBP) drug class, which includes penfluridol and fluspirilene. All three DPBPs tested (pimozide, penfluridol, and fluspirilene) improved glycemia in obese mice. While the canonical target of the DPBPs is the dopamine 2 receptor, studies in obese mice demonstrated that acute or chronic treatment with a structurally unrelated antipsychotic dopamine 2 receptor antagonist, lurasidone, was devoid of glucose-lowering actions. We further observed that the DPBPs improved glycemia in a SCOT-dependent manner in skeletal muscle, suggesting that this older class of antipsychotic agents may have utility in being repurposed for the treatment of T2D.

TRIM35-mediated degradation of nuclear PKM2 destabilizes GATA4/6 and induces P53 in cardiomyocytes to promote heart failure.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that translocates to the nucleus to regulate transcription factors in different tissues or pathologic states. Although studied extensively in cancer, its biological role in the heart remains unresolved. PKM1 is more abundant than the PKM2 isoform in cardiomyocytes, and thus, we speculated that PKM2 is not genetically redundant to PKM1 and may be critical in regulating cardiomyocyte-specific transcription factors important for cardiac survival. Here, we showed that nuclear PKM2 (S37P-PKM2) in cardiomyocytes interacts with prosurvival and proapoptotic transcription factors, including GATA4, GATA6, and P53. Cardiomyocyte-specific PKM2-deficient mice (Pkm2 Mut Cre+) developed age-dependent dilated cardiac dysfunction and had decreased amounts of GATA4 and GATA6 (GATA4/6) but increased amounts of P53 compared to Control Cre+ hearts. Nuclear PKM2 prevented caspase-1-dependent cleavage and degradation of GATA4/6 while also providing a molecular platform for MDM2-mediated reduction of P53. In a preclinical heart failure mouse model, nuclear PKM2 and GATA4/6 were decreased, whereas P53 was increased in cardiomyocytes. Loss of nuclear PKM2 was ubiquitination dependent and associated with the induction of the E3 ubiquitin ligase TRIM35. In mice, cardiomyocyte-specific TRIM35 overexpression resulted in decreased S37P-PKM2 and GATA4/6 along with increased P53 in cardiomyocytes compared to littermate controls and similar cardiac dysfunction to Pkm2 Mut Cre+ mice. In patients with dilated left ventricles, increase in TRIM35 was associated with decreased S37P-PKM2 and GATA4/6 and increased P53. This study supports a previously unrecognized role for PKM2 as a molecular platform that mediates cell signaling events essential for cardiac survival.

Stimulating myocardial pyruvate dehydrogenase activity fails to alleviate cardiac abnormalities in a mouse model of human Barth syndrome.

Barth syndrome (BTHS) is a rare genetic disorder due to mutations in the TAFAZZIN gene, leading to impaired maturation of cardiolipin and thereby adversely affecting mitochondrial function and energy metabolism, often resulting in cardiomyopathy. In a murine model of BTHS involving short-hairpin RNA mediated knockdown of Tafazzin (TazKD mice), myocardial glucose oxidation rates were markedly reduced, likely secondary to an impairment in the activity of pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. Furthermore, TazKD mice exhibited cardiac hypertrophy with minimal cardiac dysfunction. Because the stimulation of myocardial glucose oxidation has been shown to alleviate diabetic cardiomyopathy and heart failure, we hypothesized that stimulating PDH activity would alleviate the cardiac hypertrophy present in TazKD mice. In order to address our hypothesis, 6-week-old male TazKD mice and their wild-type (WT) littermates were treated with dichloroacetate (DCA; 70 mM in the drinking water), which stimulates PDH activity via inhibiting PDH kinase to prevent inhibitory phosphorylation of PDH. We utilized ultrasound echocardiography to assess cardiac function and left ventricular wall structure in all mice prior to and following 6-weeks of treatment. Consistent with systemic activation of PDH and glucose oxidation, DCA treatment improved glycemia in both TazKD mice and their WT littermates, and decreased PDH phosphorylation equivalently at all 3 of its inhibitory sites (serine 293/300/232). However, DCA treatment had no impact on left ventricular structure, or systolic and diastolic function in TazKD mice. Therefore, it is unlikely that stimulating glucose oxidation is a viable target to improve BTHS-related cardiomyopathy.

An isoproteic cocoa butter-based ketogenic diet fails to improve glucose homeostasis and promote weight loss in obese mice.

High-fat and very low-carbohydrate based ketogenic diets have gained considerable popularity as a nonpharmacological strategy for obesity, due to their potential to enhance weight loss and improve glucose homeostasis. However, the effectiveness of a ketogenic diet toward metabolic health is equivocal. To better understand the impact of ketogenic diets in obesity, male and female mice were fed a 60% cocoa butter-based high-fat diet for 16-wk to induce obesity, following which mice were transitioned to either an 85% cocoa butter fat-based ketogenic diet, a 10% cocoa butter fat-based low-fat diet, or maintained on a high-fat diet for an additional 8-wk. All experimental diets were matched for sucrose and protein content and contained an identical micronutrient profile, with complex carbohydrates being the primary carbohydrate source in the low-fat diet. The transition to a ketogenic diet was ineffective at promoting significant body fat loss and improving glucose homeostasis in obese male and female mice. Alternatively, obese male and female mice transitioned to a low-fat and high-complex carbohydrate diet exhibited beneficial body composition changes and improved glucose tolerance that may, in part, be attributed to a mild decrease in food intake and a mild increase in energy expenditure. Our findings support the consumption of a diet low in saturated fat and rich in complex carbohydrates as a potential dietary intervention for the treatment of obesity and obesity-induced impairments in glycemia. Furthermore, our results suggest that careful consideration should be taken when considering a ketogenic diet as a nonpharmacological strategy for obesity.NEW & NOTEWORTHY It has been demonstrated that ketogenic diets may be a nutritional strategy for alleviating hyperglycemia and promoting weight loss in obesity. However, there are a number of inconsistencies with many of these studies, especially with regard to the macronutrient and micronutrient compositions of the diets being compared. Our work demonstrates that a ketogenic diet that is both micronutrient-matched and isoproteic with its comparator diets fails to improve glycemia or promote weight loss in obese mice.

Pulmonary and Neurologic Effects of Mesenchymal Stromal Cell Extracellular Vesicles in a Multifactorial Lung Injury Model.

Rationale: Bronchopulmonary dysplasia, a chronic respiratory condition originating from preterm birth, is associated with abnormal neurodevelopment. Currently, there is an absence of effective therapies for bronchopulmonary dysplasia and its associated brain injury. In preclinical trials, mesenchymal stromal cell therapies demonstrate promise as a therapeutic alternative for bronchopulmonary dysplasia. Objectives: To investigate whether a multifactorial neonatal mouse model of lung injury perturbs neural progenitor cell function and to assess the ability of human umbilical cord-derived mesenchymal stromal cell extracellular vesicles to mitigate pulmonary and neurologic injury. Methods: Mice at Postnatal Day 7 or 8 were injected intraperitoneally with LPS and ventilated with 40% oxygen at Postnatal Day 9 or 10 for 8 hours. Treated animals received umbilical cord-mesenchymal stromal cell-derived extracellular vesicles intratracheally preceding ventilation. Lung morphology, vascularity, and inflammation were quantified. Neural progenitor cells were isolated from the subventricular zone and hippocampus and assessed for self-renewal, in vitro differentiation ability, and transcriptional profiles. Measurements and Main Results: The multifactorial lung injury model produced alveolar and vascular rarefaction mimicking bronchopulmonary dysplasia. Neural progenitor cells from lung injury mice showed reduced neurosphere and oligodendrocyte formation, as well as inflammatory transcriptional signatures. Mice treated with mesenchymal stromal cell extracellular vesicles showed significant improvement in lung architecture, vessel formation, and inflammatory modulation. In addition, we observed significantly increased in vitro neurosphere formation and altered neural progenitor cell transcriptional signatures. Conclusions: Our multifactorial lung injury model impairs neural progenitor cell function. Observed pulmonary and neurologic alterations are mitigated by intratracheal treatment with mesenchymal stromal cell-derived extracellular vesicles.

The anti-anginal ranolazine does not confer beneficial actions against hepatic steatosis in male mice subjected to high-fat diet and streptozotocin-induced type 2 diabetes.

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of excess fat in the liver in the absence of alcohol and increases one's risk for both diabetes and cardiovascular disease (e.g., angina). We have shown that the second-line anti-anginal therapy, ranolazine, mitigates obesity-induced NAFLD, and our aim was to determine whether these actions of ranolazine also extend to NAFLD associated with type 2 diabetes (T2D). Eight-week-old male C57BL/6J mice were fed either a low-fat diet or a high-fat diet for 15 weeks, with a single dose of streptozotocin (STZ; 75 mg/kg) administered in the high-fat diet-fed mice at 4 weeks to induce experimental T2D. Mice were treated with either vehicle control or ranolazine during the final 7 weeks (50 mg/kg once daily). We assessed glycemia via monitoring glucose tolerance, insulin tolerance, and pyruvate tolerance, whereas hepatic steatosis was assessed via quantifying triacylglycerol content. We observed that ranolazine did not improve glycemia in mice with experimental T2D, while also having no impact on hepatic triacylglycerol content. Therefore, the salutary actions of ranolazine against NAFLD may be limited to obese individuals but not those who are obese with T2D.

Nasal Septum Deviation as the Consequence of BMP-Controlled Changes to Cartilage Properties.

The nasal septum cartilage is a specialized hyaline cartilage important for normal midfacial growth. Abnormal midfacial growth is associated with midfacial hypoplasia and nasal septum deviation (NSD). However, the underlying genetics and associated functional consequences of these two anomalies are poorly understood. We have previously shown that loss of Bone Morphogenetic Protein 7 (BMP7) from neural crest (BMP7 ncko ) leads to midfacial hypoplasia and subsequent septum deviation. In this study we elucidate the cellular and molecular abnormalities underlying NSD using comparative gene expression, quantitative proteomics, and immunofluorescence analysis. We show that reduced cartilage growth and septum deviation are associated with acquisition of elastic cartilage markers and share similarities with osteoarthritis (OA) of the knee. The genetic reduction of BMP2 in BMP7 ncko mice was sufficient to rescue NSD and suppress elastic cartilage markers. To our knowledge this investigation provides the first genetic example of an in vivo cartilage fate switch showing that this is controlled by the relative balance of BMP2 and BMP7. Cellular and molecular changes similar between NSD and knee OA suggest a related etiology underlying these cartilage abnormalities.

FoxO1 inhibition alleviates type 2 diabetes-related diastolic dysfunction by increasing myocardial pyruvate dehydrogenase activity.

Type 2 diabetes (T2D) increases the risk for diabetic cardiomyopathy and is characterized by diastolic dysfunction. Myocardial forkhead box O1 (FoxO1) activity is enhanced in T2D and upregulates pyruvate dehydrogenase (PDH) kinase 4 expression, which inhibits PDH activity, the rate-limiting enzyme of glucose oxidation. Because low glucose oxidation promotes cardiac inefficiency, we hypothesize that FoxO1 inhibition mitigates diabetic cardiomyopathy by stimulating PDH activity. Tissue Doppler echocardiography demonstrates improved diastolic function, whereas myocardial PDH activity is increased in cardiac-specific FoxO1-deficient mice subjected to experimental T2D. Pharmacological inhibition of FoxO1 with AS1842856 increases glucose oxidation rates in isolated hearts from diabetic C57BL/6J mice while improving diastolic function. However, AS1842856 treatment fails to improve diastolic function in diabetic mice with a cardiac-specific FoxO1 or PDH deficiency. Our work defines a fundamental mechanism by which FoxO1 inhibition improves diastolic dysfunction, suggesting that it may be an approach to alleviate diabetic cardiomyopathy.

Barth syndrome-related cardiomyopathy is associated with a reduction in myocardial glucose oxidation.

Heart failure presents as the leading cause of infant mortality in individuals with Barth syndrome (BTHS), a rare genetic disorder due to mutations in the tafazzin (TAZ) gene affecting mitochondrial structure and function. Investigations into the perturbed bioenergetics in the BTHS heart remain limited. Hence, our objective was to identify the potential alterations in myocardial energy metabolism and molecular underpinnings that may contribute to the early cardiomyopathy and heart failure development in BTHS. Cardiac function and myocardial energy metabolism were assessed via ultrasound echocardiography and isolated working heart perfusions, respectively, in a mouse model of BTHS [doxycycline-inducible Taz knockdown (TazKD) mice]. In addition, we also performed mRNA/protein expression profiling for key regulators of energy metabolism in hearts from TazKD mice and their wild-type (WT) littermates. TazKD mice developed hypertrophic cardiomyopathy as evidenced by increased left ventricular anterior and posterior wall thickness, as well as increased cardiac myocyte cross-sectional area, though no functional impairments were observed. Glucose oxidation rates were markedly reduced in isolated working hearts from TazKD mice compared with their WT littermates in the presence of insulin, which was associated with decreased pyruvate dehydrogenase activity. Conversely, myocardial fatty acid oxidation rates were elevated in TazKD mice, whereas no differences in glycolytic flux or ketone body oxidation rates were observed. Our findings demonstrate that myocardial glucose oxidation is impaired before the development of overt cardiac dysfunction in TazKD mice, and may thus represent a pharmacological target for mitigating the development of cardiomyopathy in BTHS.NEW & NOTEWORTHY Barth syndrome (BTHS) is a rare genetic disorder due to mutations in tafazzin that is frequently associated with infantile-onset cardiomyopathy and subsequent heart failure. Although previous studies have provided evidence of perturbed myocardial energy metabolism in BTHS, actual measurements of flux are lacking. We now report a complete energy metabolism profile that quantifies flux in isolated working hearts from a murine model of BTHS, demonstrating that BTHS is associated with a reduction in glucose oxidation.

Neural crest-specific deletion of Bmp7 leads to midfacial hypoplasia, nasal airway obstruction, and disordered breathing modelling Obstructive Sleep Apnea.

Pediatric obstructive sleep apnea (OSA), a relatively common sleep-related breathing disorder (SRBD) affecting approximately 1-5% of children, is often caused by anatomical obstruction and/or collapse of the nasal and/or pharyngeal airways. The resulting sleep disruption and intermittent hypoxia lead to various systemic morbidities. Predicting the development of OSA from craniofacial features alone is currently not possible and a controversy remains if upper airway obstruction facilitates reduced midfacial growth or vice-versa. Currently, there is no rodent model that recapitulates both the development of craniofacial abnormalities and upper airway obstruction to address these questions. Here, we describe that mice with a neural crest-specific deletion of Bmp7 (Bmp7ncko) present with shorter, more acute angled cranial base, midfacial hypoplasia, nasal septum deviation, turbinate swelling and branching defects, and nasal airway obstruction. Interestingly, several of these craniofacial features develop after birth during periods of rapid midfacial growth and precede the development of an upper airway obstruction. We identified that in this rodent model, no single feature appeared to predict upper airway obstruction, but the sum of those features resulted in a reduced breathing frequency, apneas and overall reduced oxygen consumption. Metabolomics analysis of serum from peripheral blood identified increased levels of hydroxyproline, a metabolite upregulated under hypoxic conditions. As this model recapitulates many features observed in OSA, it offers unique opportunities for studying how upper airway obstruction affects breathing physiology and leads to systemic morbidities.

The GLP-1 Receptor Agonist Liraglutide Increases Myocardial Glucose Oxidation Rates via Indirect Mechanisms and Mitigates Experimental Diabetic Cardiomyopathy.

BACKGROUND: Type 2 diabetes (T2D) increases risk for cardiovascular disease. Of interest, liraglutide, a therapy for T2D that activates the glucagon-like peptide-1 receptor to augment insulin secretion, reduces cardiovascular-related death in people with T2D, though it remains unknown how liraglutide produces these actions. Notably, the glucagon-like peptide-1 receptor is not expressed in ventricular cardiac myocytes, making it likely that ventricular myocardium-independent actions are involved. We hypothesized that augmented insulin secretion may explain how liraglutide indirectly mediates cardioprotection, which thereby increases myocardial glucose oxidation. METHODS: C57BL/6J male mice were fed either a low-fat diet (lean) or were subjected to experimental T2D and treated with either saline or liraglutide 3× over a 24-hour period. Mice were subsequently euthanized and had their hearts perfused in the working mode to assess energy metabolism. A separate cohort of mice with T2D were treated with either vehicle control or liraglutide for 2 weeks for the assessment of cardiac function via ultrasound echocardiography. RESULTS: Treatment of lean mice with liraglutide increased myocardial glucose oxidation without affecting glycolysis. Conversely, direct treatment of the isolated working heart with liraglutide had no effect on glucose oxidation. These findings were recapitulated in mice with T2D and associated with increased circulating insulin levels. Furthermore, liraglutide treatment alleviated diastolic dysfunction in mice with T2D, which was associated with enhanced pyruvate dehydrogenase activity, the rate-limiting enzyme of glucose oxidation. CONCLUSIONS: Our data demonstrate that liraglutide augments myocardial glucose oxidation via indirect mechanisms, which may contribute to how liraglutide improves cardiovascular outcomes in people with T2D.

Mesenchymal Bmp7 Controls Onset of Tooth Mineralization: A Novel Way to Regulate Molar Cusp Shape.

Investigating the molecular basis for tooth shape variation provides an important glimpse into the evolution of tooth function. We recently showed that loss of mesenchymal BMP7 is sufficient to alter morphology and function of the toothrow. Here we report on the underlying mechanism. Expression of mesenchymal Bmp7 is observed at sites where mineralization is initiated, in tooth cusps of developing molars. Neural crest-specific deletion of Bmp7 (Bmp7ncko) resulted in a complete lack of dentin/enamel formation at birth, the time when mineralization is normally initiated in the upper molars, similar to what was observed in Bmp2ncko mice. Unlike loss of Bmp2, loss of Bmp7 did not affect odontoblast polarization and did not significantly alter the levels of pSmad1/5/8, but almost completely abolished canonical Wnt signaling in (pre)-ameloblasts. Tooth mineralization resumed with a 48-h delay allowing for additional mesenchymal proliferation. Enamel volume was still reduced at P4 and P8, but was comparable in erupted teeth, which were broader and had altered cusp shapes. Tooth eruption was also delayed. Overall, enamel appeared inconspicuous, although some structural changes along with reduced mineral density could be observed. Loss of Bmp7 led to an increase in mesenchymal Bmp6 suggesting an interplay between Bmp6 and Bmp7 in the regulation of mineralization initiation. Our findings show that regulation of the onset of tooth mineralization is a hitherto unsuspected mechanism controlling tooth shape variation. Initiation of tooth mineralization is regulated by a complex epithelial-mesenchymal Bmp/Wnt-signaling network to which Bmp7 contributes. This network is separate and independent of the Bmp2-signaling network regulating odontoblast cell polarization. From an evolutionary perspective, addition of Bmp7 as initiator of tooth mineralization might be akin to an upgrade of an existing computer operating system. While not essential, it provides obviously sufficient advantage warranting its evolutionary incorporation.

Pimozide Alleviates Hyperglycemia in Diet-Induced Obesity by Inhibiting Skeletal Muscle Ketone Oxidation.

Perturbations in carbohydrate, lipid, and protein metabolism contribute to obesity-induced type 2 diabetes (T2D), though whether alterations in ketone body metabolism influence T2D pathology is unknown. We report here that activity of the rate-limiting enzyme for ketone body oxidation, succinyl-CoA:3-ketoacid-CoA transferase (SCOT/Oxct1), is increased in muscles of obese mice. We also found that the diphenylbutylpiperidine pimozide, which is approved to suppress tics in individuals with Tourette syndrome, is a SCOT antagonist. Pimozide treatment reversed obesity-induced hyperglycemia in mice, which was phenocopied in mice with muscle-specific Oxct1/SCOT deficiency. These actions were dependent on pyruvate dehydrogenase (PDH/Pdha1) activity, the rate-limiting enzyme of glucose oxidation, as pimozide failed to alleviate hyperglycemia in obese mice with a muscle-specific Pdha1/PDH deficiency. This work defines a fundamental contribution of enhanced ketone body oxidation to the pathology of obesity-induced T2D, while suggesting pharmacological SCOT inhibition as a new class of anti-diabetes therapy.

Late Rescue Therapy with Cord-Derived Mesenchymal Stromal Cells for Established Lung Injury in Experimental Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), the main complication of extreme prematurity, has lifelong consequences for lung health. Mesenchymal stromal cells (MSCs) prevent lung injury in experimental BPD in newborn rodents when given in the immediate neonatal period. Whether MSC therapy can restore normal lung growth after established lung injury in adulthood is clinically relevant, but currently unknown. Experimental BPD was achieved by exposing newborn rats to 95% O2 from postnatal days 4-14. Human umbilical cord-derived MSCs were intratracheally administered to rats (1 × 106cells/kg body weight) as a single dose at 3 or 6 months of age followed by assessment at 5 or 8 months of age, respectively. Lung alveolar structure and vessel density were histologically analyzed. O2-exposed rats exhibited persistent lung injury characterized by arrested alveolar growth with airspace enlargement and a lower vessel density at both 5 and 8 months of age compared with controls. Single-dose MSC treatment at 3 months partially attenuated O2-induced alveolar injury and restored vessel density at 5 months. Treatment with a single dose at 6 months did not attenuate alveolar injury or vessel density at 8 months. However, treatment with multiple MSC doses at 6, 6.5, 7, and 7.5 months significantly attenuated alveolar injury and improved vessel density at 8 months of age. Treatment of the adult BPD lung with MSCs has the potential to improve lung injury if administered in multiple doses or at an early stage of adulthood.

l-Citrulline supplementation improves glucose and exercise tolerance in obese male mice.

NEW FINDINGS: What is the central question of the study? Does the action of l-citrulline, which has been shown to augment performance in animals and athletes, possibly via increasing mitochondrial function, translate to obese animals, and does this improve glycaemia? What is the main finding and its importance? Chronic supplementation with l-citrulline improves not only exercise capacity, but also glycaemia in obese mice, which would be beneficial as obese individuals are at increased risk for type 2 diabetes. However, l-citrulline supplementation also caused a mild impairment in insulin signalling and insulin tolerance in obese mice. ABSTRACT: l-Citrulline is an organic α-amino acid that has been shown to have a number of salutary actions on whole-body physiology, including reducing muscle wasting and augmenting exercise and muscle performance. The latter has been suggested to arise from elevations in mitochondrial function. Because enhancing mitochondrial function has been proposed as a novel strategy to mitigate insulin resistance, our goal was to determine whether supplementation with l-citrulline could also improve glycaemia in an experimental mouse model of obesity. We hypothesized that l-citrulline treatment would improve glycaemia in obese mice, and this would be associated with elevations in skeletal muscle mitochondrial function. Ten-week-old C57BL/6J mice were fed either a low-fat (10% kcal from lard) or a high-fat (60% kcal from lard) diet, while receiving drinking water supplemented with either vehicle or l-citrulline (0.6 g l-1 ) for 15 weeks. Glucose homeostasis was assessed via glucose/insulin tolerance testing, while in vivo metabolism was assessed via indirect calorimetry, and forced exercise treadmill testing was utilized to assess endurance. As expected, obese mice supplemented with l-citrulline exhibited an increase in exercise capacity, which was associated with an improvement in glucose tolerance. Consistent with augmented mitochondrial function, we observed an increase in whole body oxygen consumption rates in obese mice supplemented with l-citrulline. Surprisingly, l-citrulline supplementation worsened insulin tolerance and reduced insulin signalling in obese mice. Taken together, although l-citrulline supplementation improves both glucose tolerance and exercise capacity in obese mice, caution must be applied with its broad use as a nutraceutical due to a potential deterioration of insulin sensitivity.