Acetyl-CoA Carboxylase Inhibition Improves Multiple Dimensions of NASH Pathogenesis in Model Systems.

BACKGROUND & AIMS: Disordered metabolism, steatosis, hepatic inflammation, and fibrosis contribute to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase (ACC) catalyzes the first committed step in de novo lipogenesis (DNL) and modulates mitochondrial fatty acid oxidation. Increased hepatic DNL flux and reduced fatty acid oxidation are hypothesized to contribute to steatosis. Some proinflammatory cells also show increased dependency on DNL, suggesting that ACC may regulate aspects of the inflammatory response in NASH. PF-05221304 is an orally bioavailable, liver-directed ACC1/2 inhibitor. The present studies sought to evaluate the effects of PF-05221304 on NASH pathogenic factors in experimental model systems. METHODS: The effects of PF-05221304 on lipid metabolism, steatosis, inflammation, and fibrogenesis were investigated in both primary human-derived in vitro systems and in vivo rodent models. RESULTS: PF-05221304 inhibited DNL, stimulated fatty acid oxidation, and reduced triglyceride accumulation in primary human hepatocytes, and reduced DNL and steatosis in Western diet-fed rats in vivo, showing the potential to reduce hepatic lipid accumulation and potentially lipotoxicity. PF-05221304 blocked polarization of human T cells to proinflammatory but not anti-inflammatory T cells, and suppressed activation of primary human stellate cells to myofibroblasts in vitro, showing direct effects on inflammation and fibrogenesis. Consistent with these observations, PF-05221304 also reduced markers of inflammation and fibrosis in the diethylnitrosamine chemical-induced liver injury model and the choline-deficient, high-fat-fed rat model. CONCLUSIONS: The liver-directed dual ACC1/ACC2 inhibitor directly improved multiple nonalcoholic fatty liver disease/NASH pathogenic factors including steatosis, inflammation, and fibrosis in both human-derived in vitro systems and rat models.

Structure-function relationships of the soluble form of the antiaging protein Klotho have therapeutic implications for managing kidney disease.

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and ß-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

No Evidence of Neurogenesis in Adult Rat Sympathetic Ganglia Following Guanethidine-Induced Neuronal Loss.

The potential for neurogenesis in the cranial (superior) cervical ganglia (SCG) of the sympathetic nervous system was evaluated. Eleven consecutive daily doses of guanethidine (100 mg/kg/d) were administered intraperitoneally to rats in order to destroy postganglionic sympathetic neurons in SCG. Following the last dose, animals were allowed to recover 1, 3, or 6 months. Right and left SCG from guanethidine-treated and age-matched, vehicle-treated control rats were harvested for histopathologic, morphometric, and stereologic evaluations. Both morphometric and stereologic evaluations confirmed neuron loss following guanethidine treatment. Morphometric analysis revealed a 50% to 60% lower number of tyrosine hydroxylase (TH)-positive neurons per unit area of SCG at both 3 and 6 months of recovery, compared to ganglia of age-matched controls, with no evidence of restoration of neuron density between 3 and 6 months. Reductions in TH-positive neurons following guanethidine treatment were corroborated by unbiased stereology of total hematoxylin and eosin-stained neuron numbers in SCG. Stereologic analyses revealed that total neuron counts were lower by 37% at 3 months of recovery when compared to age-matched vehicle controls, again with no obvious restoration between 3 and 6 months. Thus, no evidence was found that postganglionic neurons of the sympathetic nervous system in the adult rat have a neurogenic capacity.

Myeloperoxidase inhibition in mice alters atherosclerotic lesion composition.

Myeloperoxidase (MPO) is a highly abundant protein within the neutrophil that is associated with lipoprotein oxidation, and increased plasma MPO levels are correlated with poor prognosis after myocardial infarct. Thus, MPO inhibitors have been developed for the treatment of heart failure and acute coronary syndrome in humans. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide PF-06282999 is a recently described selective small molecule mechanism-based inactivator of MPO. Here, utilizing PF-06282999, we investigated the role of MPO to regulate atherosclerotic lesion formation and composition in the Ldlr-/- mouse model of atherosclerosis. Though MPO inhibition did not affect lesion area in Ldlr-/- mice fed a Western diet, reduced necrotic core area was observed in aortic root sections after MPO inhibitor treatment. MPO inhibition did not alter macrophage content in and leukocyte homing to atherosclerotic plaques. To assess non-invasive monitoring of plaque inflammation, [18F]-Fluoro-deoxy-glucose (FDG) was administered to Ldlr-/- mice with established atherosclerosis that had been treated with clinically relevant doses of PF-06282999, and reduced FDG signal was observed in animals treated with a dose of PF-06282999 that corresponded with reduced necrotic core area. These data suggest that MPO inhibition does not alter atherosclerotic plaque area or leukocyte homing, but rather alters the inflammatory tone of atherosclerotic lesions; thus, MPO inhibition could have utility to promote atherosclerotic lesion stabilization and prevent atherosclerotic plaque rupture.

From the Cover: Fenretinide, Troglitazone, and Elmiron Add to Weight of Evidence Support for Hemangiosarcoma Mode-of-Action From Studies in Mice.

Pharmaceuticals and chemicals produce hemangiosarcomas (HS) in mice, often by nongenotoxic, proliferative mechanisms. A mode-of-action (MOA) for hemangiosarcoma was proposed based on information presented at an international workshop (Cohen et al., Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18.). Five key elements of the MOA were articulated and included hypoxia, macrophage activation, increased angiogenic growth factors, dysregulated angiogenesis/erythropoiesis, and endothial cell proliferation. The goal of the current study was to add to the weight-of-evidence for the proposed MOA by assessing these key elements with 3 different compounds of varying potency for HS induction: fenretinide (high), troglitazone (intermediate), and elmiron (low). Multiple endpoints, including hypoxia (hyproxyprobe, transcriptomics), endothelial cell (EC) proliferation, and clinical and anatomic pathology, were assessed after 2, 4, and 13-weeks of treatment in B6C3F1 mice. All 3 compounds demonstrated strong evidence for dysregulated erythropoiesis (decrease in RBC and a failure to increase reticulocytes) and macrophage activation (4- to 11-fold increases); this pattern of hematological changes in mice might serve as an early biomarker to evaluate EC proliferation in suspected target organs for potential HS formation. Fenretinide demonstrated all 5 key elements, while troglitazone demonstrated 4 and elmiron demonstrated 3. Transcriptomics provided support for the 5 elements of the MOA, but was not any more sensitive than hypoxyprobe immunohistochemistry for detecting hypoxia. The overall transcriptional evidence for the key elements of the proposed MOA was also consistent with the potency of HS induction. These data, coupled with the previous work with 2-butoxyethanol and pregablin, increase the weight-of-evidence for the proposed MOA for HS formation.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

Selective Activation of AMPK ß1-Containing Isoforms Improves Kidney Function in a Rat Model of Diabetic Nephropathy.

Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.

Neurophysiological assessment of sympathetic cardiovascular activity after loss of postganglionic neurons in the anesthetized rat.

The goal of this study was to determine the degree of sympathetic postganglionic neuronal loss required to impair cardiovascular-related sympathetic activity. To produce neuronal loss separate groups of rats were treated daily with guanethidine for either 5days or 11days, followed by a recovery period. Sympathetic activity was measured by renal sympathetic nerve activity (RSNA). Stereology of thoracic (T13) ganglia was performed to determine neuronal loss. Despite loss of more than two thirds of neurons in T13 ganglia in both treated groups no effect on resting blood pressure (BP) or heart rate (HR) was detected. Basal RSNA in rats treated for 5days (0.61±0.10µV∗s) and 11days (0.37±0.08µV∗s) was significantly less than vehicle-treated rats (0.99±0.13µV∗s, p<0.05). Increases in RSNA by baroreceptor unloading were significantly lower in 5-day (1.09±0.19µV∗s) and 11-day treated rats (0.59±0.11µV∗s) compared with vehicle-treated rats (1.82±0.19µV∗s, p<0.05). Increases in RSNA to chemoreceptor stimulation were significantly lower in 5-day treated rats (1.54±0.25µV∗s) compared with vehicle-treated rats (2.69±0.23µV∗s, p<0.05). Increases in RSNA in 11-day treated rats were significantly lower (0.75±0.15µV∗s, p<0.05) compared with both vehicle-treated and 5-day treated rats. A positive correlation of neurons to sympathetic responsiveness but not basal activity was detected. These data suggest that diminished capacity for reflex sympathetic responsiveness rather than basal activity alone must be assessed for complete detection of neurophysiological cardiovascular impairment.

High throughput object-based image analysis of ß-amyloid plaques in human and transgenic mouse brain.

Advances in imaging technology have enabled automated approaches for quantitative image analysis. In this study, a high content object based image analysis method was developed for quantification of ß-amyloid (Aß) plaques in postmortem brains of Alzheimer's disease (AD) subjects and in transgenic mice over overexpressing Aß. Digital images acquired from immunohistochemically stained sections of the superior frontal gyrus were analyzed for Aß plaque burden using a Definiens object-based segmentation approach. Blinded evaluation of Aß stained sections from AD and aged matched human subjects accurately identified AD cases with one exception. Brains from transgenic mice overexpressing Aß (PS1APP mice) were also evaluated by our Definiens object based image analysis approach. We observed an age-dependent increase in the amount of Aß plaque load that we quantified in both the hippocampus and cortex. From the contralateral hemisphere, we measured the amount of Aß in brain homogenates biochemically and observed a significant correlation between our biochemical measurements and those that we measured by our object based Definiens system in the hippocampus. Assessment of Aß plaque load in PS1APP mice using a manual segmentation technique (Image-Pro Plus) confirmed the results of our object-based image analysis approach. Image acquisition and analysis of 32 stained human slides and 100 mouse slides were executed in 8 h and 22 h, respectively supporting the relatively high throughput features of the Definiens platform. The data show that digital imaging combined with object based image analysis is a reliable and efficient approach to quantifying Aß plaques in human and mouse brain.

Postnatal PPARdelta activation and myostatin inhibition exert distinct yet complimentary effects on the metabolic profile of obese insulin-resistant mice.

BACKGROUND: Interventions for T2DM have in part aimed to mimic exercise. Here, we have compared the independent and combined effects of a PPARdelta agonist and endurance training mimetic (GW501516) and a myostatin antibody and resistance training mimetic (PF-879) on metabolic and performance outcomes in obese insulin resistant mice. METHODOLOGY/PRINCIPAL FINDINGS: Male ob/ob mice were treated for 6 weeks with vehicle, GW501516, PF-879, or GW501516 in combination with PF-879. The effects of the interventions on body composition, glucose homeostasis, glucose tolerance, energy expenditure, exercise capacity and metabolic gene expression were compared at the end of study. GW501516 attenuated body weight and fat mass accumulation and increased the expression of genes of oxidative metabolism. In contrast, PF-879 increased body weight by driving muscle growth and altered the expression of genes involved in insulin signaling and glucose metabolism. Despite their differences, both interventions alone improved glucose homeostasis. Moreover, GW501516 more effectively improved serum lipids, and PF-879 uniquely increased energy expenditure, exercise capacity and adiponectin levels. When combined the robust effects of GW501516 and/or PF-879 on body weight, adiposity, muscle mass, glycemia, serum lipids, energy expenditure and exercise capacity were highly conserved. CONCLUSIONS/SIGNIFICANCE: The data, for the first time, demonstrate postnatal inhibition of myostatin not only promotes gains in muscle mass similar to resistance training,but improves metabolic homeostasis. In several instances, these effects were either distinct from or complimentary to those of GW501516. The data further suggest that strategies to increase muscle mass, and not necessarily oxidative capacity, may effectively counter insulin resistance and T2DM.

Investigating cytoskeletal alterations as a potential marker of retinal and lens drug-related toxicity.

Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin fluorescence at noncytotoxic concentrations in all cells tested as observed by confocal microscopy. Developing an assay that predicts ocular toxicity helps the development process by prioritizing compounds for further investigation. Drug-induced cytoskeletal alterations may be useful as a potential safety-screening marker of retinal and lens toxicity. The knowledge of actin molecular biology and the application of other mechanistic screens to toxicology are discussed. Reducing this work to a high-throughput platform will enable chemists to select compounds with a reduced risk of ocular toxicity.

Pentamidine reduces hERG expression to prolong the QT interval.

Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC50 of 252 microM. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 microM, with a slight prolongation of the action potential duration at this concentration. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (approximately 1 microM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 microM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-microM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.