Pan AMPK Activation Protects Tubules in Rat Ischemic Acute Kidney Injury.

Acute Kidney Injury (AKI) is characterized by an abrupt decline in kidney function and has been associated with excess risks of death, kidney disease progression, and cardiovascular events. The kidney has a high energetic demand with mitochondrial health being essential to renal function and damaged mitochondria has been reported across AKI subtypes. 5' adenosine monophosphate-activated protein kinase (AMPK) activation preserves cellular energetics through improvement of mitochondrial function and biogenesis when ATP levels are low such as under ischemia-induced AKI. We developed a selective potent small molecule pan AMPK activator, compound 1, and tested its ability to increase AMPK activity and preserve kidney function during ischemia/reperfusion injury in rats. A single administration of 1 caused sustained activation of AMPK for at least 24 hours, protected against acute tubular necrosis, and reduced clinical markers of tubular injury such as NephroCheck and Fractional Excretion of Sodium (FENa). Reduction in plasma creatinine and increased Glomerular Filtration Rate (GFR) indicated preservation of kidney function. Surprisingly, we observed a strong diuretic effect of AMPK activation associated with natriuresis both with and without AKI. Our findings demonstrate that activation of AMPK leads to protection of tubular function under hypoxic/ischemic conditions which holds promise as a potential novel therapeutic approach for AKI. Significance Statement No approved pharmacological therapies currently exist for acute kidney injury. We developed Compound 1 which dose-dependently activated AMPK in the kidney and protected kidney function and tubules after ischemic renal injury in the rat. This was accompanied by natriuresis in injured as well as uninjured rats.

Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

GPR120 suppresses adipose tissue lipolysis and synergizes with GPR40 in antidiabetic efficacy.

GPR40 and GPR120 are fatty acid sensors that play important roles in glucose and energy homeostasis. GPR40 potentiates glucose-dependent insulin secretion and demonstrated in clinical studies robust glucose lowering in type 2 diabetes. GPR120 improves insulin sensitivity in rodents, albeit its mechanism of action is not fully understood. Here, we postulated that the antidiabetic efficacy of GPR40 could be enhanced by coactivating GPR120. A combination of GPR40 and GPR120 agonists in db/db mice, as well as a single molecule with dual agonist activities, achieved superior glycemic control compared with either monotherapy. Compared with a GPR40 selective agonist, the dual agonist improved insulin sensitivity in ob/ob mice measured by hyperinsulinemic-euglycemic clamp, preserved islet morphology, and increased expression of several key lipolytic genes in adipose tissue of Zucker diabetic fatty rats. Novel insights into the mechanism of action for GPR120 were obtained. Selective GPR120 activation suppressed lipolysis in primary white adipocytes, although this effect was attenuated in adipocytes from obese rats and obese rhesus, and sensitized the antilipolytic effect of insulin in rat and rhesus primary adipocytes. In conclusion, GPR120 agonism enhances insulin action in adipose tissue and yields a synergistic efficacy when combined with GPR40 agonism.

Physiological Expression of AMPKγ2RG Mutation Causes Wolff-Parkinson-White Syndrome and Induces Kidney Injury in Mice.

Mutations of the AMP-activated kinase gamma 2 subunit (AMPKγ2), N488I (AMPKγ2NI) and R531G (AMPKγ2RG), are associated with Wolff-Parkinson-White (WPW) syndrome, a cardiac disorder characterized by ventricular pre-excitation in humans. Cardiac-specific transgenic overexpression of human AMPKγ2NI or AMPKγ2RG leads to constitutive AMPK activation and the WPW phenotype in mice. However, overexpression of these mutant proteins also caused profound, non-physiological increase in cardiac glycogen, which might abnormally alter the true phenotype. To investigate whether physiological levels of AMPKγ2NI or AMPKγ2RG mutation cause WPW syndrome and metabolic changes in other organs, we generated two knock-in mouse lines on the C57BL/6N background harboring mutations of human AMPKγ2NI and AMPKγ2RG, respectively. Similar to the reported phenotypes of mice overexpressing AMPKγ2NI or AMPKγ2RG in the heart, both lines developed WPW syndrome and cardiac hypertrophy; however, these effects were independent of cardiac glycogen accumulation. Compared with AMPKγ2WT mice, AMPKγ2NI and AMPKγ2RG mice exhibited reduced body weight, fat mass, and liver steatosis when fed with a high fat diet (HFD). Surprisingly, AMPKγ2RG but not AMPKγ2NI mice fed with an HFD exhibited severe kidney injury characterized by glycogen accumulation, inflammation, apoptosis, cyst formation, and impaired renal function. These results demonstrate that expression of AMPKγ2NI and AMPKγ2RG mutations at physiological levels can induce beneficial metabolic effects but that this is accompanied by WPW syndrome. Our data also reveal an unexpected effect of AMPKγ2RG in the kidney, linking lifelong constitutive activation of AMPK to a potential risk for kidney dysfunction in the context of an HFD.

Phenotyping of adipose, liver, and skeletal muscle insulin resistance and response to pioglitazone in spontaneously obese rhesus monkeys.

Insulin resistance and diabetes can develop spontaneously with obesity and aging in rhesus monkeys, highly similar to the natural history of obesity, insulin resistance, and progression to type 2 diabetes in humans. The current studies in obese rhesus were undertaken to assess hepatic and adipose contributions to systemic insulin resistance-currently, a gap in our knowledge-and to benchmark the responses to pioglitazone (PIO). A two-step hyperinsulinemic-euglycemic clamp, with tracer-based glucose flux estimates, was used to measure insulin resistance, and in an intervention study was repeated following 6 wk of PIO treatment (3 mg/kg). Compared with lean healthy rhesus, obese rhesus has a 60% reduction of glucose utilization during a high insulin infusion and markedly impaired suppression of lipolysis, which was evident at both low and high insulin infusion. However, obese dysmetabolic rhesus manifests only mild hepatic insulin resistance. Six-week PIO treatment significantly improved skeletal muscle and adipose insulin resistance (by ~50%). These studies strengthen the concept that insulin resistance in obese rhesus closely resembles human insulin resistance and indicate the value of obese rhesus for appraising new insulin-sensitizing therapeutics.

Inhibition of Factor XIa Reduces the Frequency of Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits.

Factor XI (FXI) is an integral component of the intrinsic pathway of the coagulation cascade and plays a critical role in thrombus formation. Because its role in the pathogenesis of cerebral microembolic signals (MES) is unclear, this study used a potent and selective small molecule inhibitor of FXIa, compound 1, to assess the effect of FXI blockade in our recently established preclinical model of cerebral MES induced by FeCl3 injury of the carotid artery in male New Zealand White rabbits. Ascending doses of compound 1 were evaluated simultaneously for both carotid arterial thrombosis by a Doppler flowmeter and MES in the middle cerebral artery by a transcranial Doppler. Plasma drug exposure and pharmacodynamic responses to compound 1 treatment were also assessed. The effective dose for 50% inhibition (ED50) of thrombus formation was 0.003 mg/kg/h compound 1, i.v. for the integrated blood flow, 0.004 mg/kg/h for reduction in thrombus weight, and 0.106 mg/kg/h for prevention of MES. The highest dose, 3 mg/kg/h compound 1, achieved complete inhibition in both thrombus formation and MES. In addition, we assessed the potential bleeding liability of compound 1 (5 mg/kg/h, i.v., >1250-fold ED50 levels in arterial thrombosis) in rabbits using a cuticle bleeding model, and observed about 2-fold (not statistically significant) prolongation in bleeding time. Our study demonstrates that compound 1 produced a robust and dose-dependent inhibition of both arterial thrombosis and MES, suggesting that FXIa blockade may represent a novel therapeutic strategy for the reduction in MES in patients at risk for ischemic stroke.

Enhancing Studies of Pharmacodynamic Mechanisms via Measurements of Metabolic Flux: Fundamental Concepts and Guiding Principles for Using Stable Isotope Tracers.

Drug discovery and development efforts are largely based around a common expectation, namely, that direct or indirect action on a cellular process (e.g., statin-mediated enzyme inhibition or insulin-stimulated receptor activation) will have a beneficial impact on physiologic homeostasis. To expand on this, one could argue that virtually all pharmacologic interventions attempt to influence the flow of "traffic" in a biochemical network, irrespective of disease or modality. Since stable isotope tracer kinetic methods provide a measure of traffic flow (i.e., metabolic flux), their inclusion in study designs can yield novel information regarding pathway biology; the application of such methods requires the integration of knowledge in physiology, analytical chemistry, and mathematical modeling. Herein, we review the fundamental concepts that surround the use of tracer kinetics, define basic terms, and outline guiding principles via theoretical and experimental problems. Specifically, one needs to 1) recognize the types of biochemical events that change isotopic enrichments, 2) appreciate the distinction between fractional turnover and flux rate, and 3) be aware of the subtle differences between tracer kinetics and pharmacokinetics. We hope investigators can use the framework presented here to develop applications that address their specific questions surrounding biochemical flux, and thereby gain insight into the pathophysiology of disease states, and examine pharmacodynamic mechanisms.

Quantifying rates of glucose production in vivo following an intraperitoneal tracer bolus.

Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a "glucose production index" by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.

Glucagon receptor antagonism induces increased cholesterol absorption.

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

The mitochondrial fission protein Drp1 in liver is required to mitigate NASH and prevents the activation of the mitochondrial ISR.

OBJECTIVE: The mitochondrial fission protein Drp1 was proposed to promote NAFLD, as inhibition of hepatocyte Drp1 early in life prevents liver steatosis induced by high-fat diet in mice. However, whether Drp1-knockdown in older mice can reverse established NASH is unknown. METHODS: N-acetylgalactosamine-siRNA conjugates, an FDA approved method to deliver siRNA selectively to hepatocytes, were used to knockdown hepatocyte-Drp1 in mice (NAG-Drp1si). NASH was induced in C57BL/6NTac mice by Gubra-Amylin-NASH diet (D09100310, 40% fat, 22% fructose and 2% cholesterol) and treatment with NAG-Drp1si was started at week 24 of diet. Circulating transaminases, liver histology, gene expression of fibrosis and inflammation markers, and hydroxyproline synthesis determined NASH severity. Liver NEFA and triglycerides were quantified by GC/MS. Mitochondrial function was determined by respirometry. Western blots of Oma1, Opa1, p-eIf2α, as well as transcriptional analyses of Atf4-regulated genes determined ISR engagement. RESULTS: NAG-Drp1si treatment decreased body weight and induced liver inflammation in adult healthy mice. Increased hepatic Gdf15 production was the major contributor to body-weight loss caused by NAG-Drp1si treatment, as Gdf15 receptor deletion (Gfral KO) prevented the decrease in food intake and mitigated weight loss. NAG-Drp1si activated the Atf4-controlled integrated stress response (ISR) to increase hepatic Gdf15 expression. NAG-Drp1si in healthy mice caused ER stress and activated the mitochondrial protease Oma1, which are the ER and mitochondrial triggers that activate the Atf4-controlled ISR. Remarkably, induction of NASH was not sufficient to activate Oma1 in liver. However, NAG-Drp1si treatment was sufficient to activate Oma1 in adult mice with NASH, as well as exacerbating NASH-induced ER stress. Consequently, NAG-Drp1si treatment in mice with NASH led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis. CONCLUSION: Drp1 mitigates NASH by decreasing ER stress, preventing Oma1 activation and ISR exacerbation. The elevation in Gdf15 actions induced by NAG-Drp1si might represent an adaptive response decreasing the nutrient load to liver when mitochondria are misfunctional. Our study argues against blocking Drp1 in hepatocytes to combat NASH.

Discovery of MK-1462: GLP-1 and Glucagon Receptor Dual Agonist for the Treatment of Obesity and Diabetes.

Peptide-based analogues of the gut-derived incretin hormone, glucagon-like peptide 1 (GLP1), stimulate insulin secretion in a glucose-dependent manner. Currently marketed GLP1 receptor (GLP1R) agonists are safe and effective in the management of Type 2 diabetes but often offer only modest weight loss. This has prompted the search for safe and effective alternatives to enhance the weight loss component of these treatments. We have demonstrated that concomitant activation GLP1R and the glucagon receptor (GCGR) can improve glucose metabolism and provide superior weight loss when compared to selective GLP1R agonism in preclinical species. This paper will highlight chemistry structure-activity relationship optimization and summarize in vivo efficacy studies toward the discovery of a once daily balanced dual agonist 12 (MK-1462), which was advanced into clinical trials.

Functionally selective signaling and broad metabolic benefits by novel insulin receptor partial agonists.

Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.

Discovery of Insulin Receptor Partial Agonists MK-5160 and MK-1092 as Novel Basal Insulins with Potential to Improve Therapeutic Index.

We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of ∼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.

Use of a QM/MM-based FEP method to evaluate the anomalous hydration behavior of simple alkyl amines and amides: application to the design of FBPase inhibitors for the treatment of type-2 diabetes.

Standard molecular mechanics (MM) force fields predict a nearly linear decrease in hydration free energy with each successive addition of a methyl group to ammonia or acetamide, whereas a nonadditive relationship is observed experimentally. In contrast, the non-additive hydration behavior is reproduced directly using a quantum mechanics (QM)/MM-based free-energy perturbation (FEP) method wherein the solute partial atomic charges are updated at every window. Decomposing the free energies into electrostatic and van der Waals contributions and comparing the results with the corresponding free energies obtained using a conventional FEP method and a QM/MM method wherein the charges are not updated suggests that inaccuracies in the electrostatic free energies are the primary reason for the inability of the conventional FEP method to predict the experimental findings. The QM/MM-based FEP method was subsequently used to evaluate inhibitors of the diabetes drug target fructose-1,6-bisphosphatase adenosine 5'-monophosphate and 6-methylamino purine riboside 5'-monophosphate. The predicted relative binding free energy was consistent with the experimental findings, whereas the relative binding free energy predicted using the conventional FEP method differed from the experimental finding by an amount consistent with the overestimated relative solvation free energies calculated for alkylamines. Accordingly, the QM/MM-based FEP method offers potential advantages over conventional FEP methods, including greater accuracy and reduced user input. Moreover, since drug candidates often contain either functionality that is inadequately treated by MM (e.g., simple alkylamines and alkylamides) or new molecular scaffolds that require time-consuming development of MM parameters, these advantages could enable future automation of FEP calculations as well as greatly increase the use and impact of FEP calculations in drug discovery.

Antiviral efficacy upon administration of a HepDirect prodrug of 2'-C-methylcytidine to hepatitis C virus-infected chimpanzees.

Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide, and the current standard of care, a combination of pegylated interferon alpha and ribavirin, is efficacious in achieving sustained viral response in ~50% of treated patients. Novel therapies under investigation include the use of nucleoside analog inhibitors of the viral RNA-dependent RNA polymerase. NM283, a 3'-valyl ester prodrug of 2'-C-methylcytidine, has demonstrated antiviral efficacy in HCV-infected patients (N. Afdhal et al., J. Hepatol. 46[Suppl. 1]:S5, 2007; N. Afdhal et al., J. Hepatol. 44[Suppl. 2]:S19, 2006). One approach to increase the antiviral efficacy of 2'-C-methylcytidine is to increase the concentration of the active inhibitory species, the 5'-triphosphate, in infected hepatocytes. HepDirect prodrug technology can increase intracellular concentrations of a nucleoside triphosphate in hepatocytes by introducing the nucleoside monophosphate into the cell, bypassing the initial kinase step that is often rate limiting. Screening for 2'-C-methylcytidine triphosphate levels in rat liver after oral dosing identified 1-[3,5-difluorophenyl]-1,3-propandiol as an efficient prodrug modification. To determine antiviral efficacy in vivo, the prodrug was administered separately via oral and intravenous dosing to two HCV-infected chimpanzees. Circulating viral loads declined by ~1.4 log(10) IU/ml and by >3.6 log(10) IU/ml after oral and intravenous dosing, respectively. The viral loads rebounded after the end of dosing to predose levels. The results indicate that a robust antiviral response can be achieved upon administration of the prodrug.

Fructose-1, 6-bisphosphatase inhibitors for reducing excessive endogenous glucose production in type 2 diabetes.

Fructose-1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis, has emerged as an important target for the treatment of type 2 diabetes due to the well-recognized role of excessive endogenous glucose production (EGP) in the hyperglycemia characteristic of the disease. Inhibitors of FBPase are expected to fulfill an unmet medical need because the majority of current antidiabetic medications act primarily on insulin resistance or insulin insufficiency and do not reduce gluconeogenesis effectively or in a direct manner. Despite significant challenges, potent and selective inhibitors of FBPase targeting the allosteric site of the enzyme were identified by means of a structure-guided design strategy that used the natural inhibitor, adenosine monophosphate (AMP), as the starting point. Oral delivery of these anionic FBPase inhibitors was enabled by a novel diamide prodrug class. Treatment of diabetic rodents with CS-917, the best characterized of these prodrugs, resulted in a reduced rate of gluconeogenesis and EGP. Of note, inhibition of gluconeogenesis by CS-917 led to the amelioration of both fasting and postprandial hyperglycemia without weight gain, incidence of hypoglycemia, or major perturbation of lactate or lipid homeostasis. Furthermore, the combination of CS-917 with representatives of the insulin sensitizer or insulin secretagogue drug classes provided enhanced glycemic control. Subsequent clinical evaluations of CS-917 revealed a favorable safety profile as well as clinically meaningful reductions in fasting glucose levels in patients with T2DM. Future trials of MB07803, a second generation FBPase inhibitor with improved pharmacokinetics, will address whether this novel class of antidiabetic agents can provide safe and long-term glycemic control.

PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells.

Non-alcoholic steatohepatitis (NASH) results, in part, from the interaction of metabolic derangements with predisposing genetic variants, leading to liver-related complications and mortality. The strongest genetic determinant is a highly prevalent missense variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3 p.I148M). In human liver hepatocytes PNPLA3 localizes to the surface of lipid droplets where the mutant form is believed to enhance lipid accumulation and release of pro-inflammatory cytokines. Less is known about the role of PNPLA3 in hepatic stellate cells (HSCs). Here we characterized HSC obtained from patients carrying the wild type (n = 8 C/C) and the heterozygous (n = 6, C/G) or homozygous (n = 6, G/G) PNPLA3 I148M and investigated the effect of genotype and PNPLA3 downregulation on baseline and TGF-ß-stimulated fibrotic gene expression. HSCs from all genotypes showed comparable baseline levels of PNPLA3 and expression of the fibrotic genes α-SMA, COL1A1, TIMP1 and SMAD7. Treatment with TGF-ß increased PNPLA3 expression in all 3 genotypes (~2-fold) and resulted in similar stimulation of the expression of several fibrogenic genes. In primary human HSCs carrying wild-type (WT) PNPLA3, siRNA treatment reduced PNPLA3 mRNA by 79% resulting in increased expression of α-SMA, Col1a1, TIMP1, and SMAD7 in cells stimulated with TGF-ß. Similarly, knock-down of PNPLA3 in HSCs carrying either C/G or G/G genotypes resulted in potentiation of TGF-ß induced expression of fibrotic genes. Knockdown of PNPLA3 did not impact fibrotic gene expression in the absence of TGF-ß treatment. Together, these data indicate that the presence of the I148M PNPLA3 mutation in HSC has no effect on baseline activation and that downregulation of PNPLA3 exacerbates the fibrotic response irrespective of the genotype.

Reduction of hepatic steatosis in rats and mice after treatment with a liver-targeted thyroid hormone receptor agonist.

UNLABELLED: Non-alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver disease, with a prevalence ranging from 10% to 30%. The use of thyroid hormone receptor (TR) agonists for the treatment of NAFLD has not been considered viable because thyroid hormones increase free fatty acid (FFA) flux from the periphery to the liver, induce hepatic lipogenesis, and therefore could potentially cause steatosis. MB07811 is an orally active HepDirect prodrug of MB07344, a liver-targeted TR-beta agonist. The purpose of these studies was to assess the effects of MB07811 on whole body and liver lipid metabolism of normal rodents and rodent models of hepatic steatosis. In the current studies, MB07811 markedly reduced hepatic steatosis as well as reduced plasma FFA and triglycerides. In contrast to MB07811, T(3) induced adipocyte lipolysis in vitro and in vivo and had a diminished ability to decrease hepatic steatosis. This suggests the influx of FFA from the periphery to the liver may partially counteract the antisteatotic activity of T(3). Clearance of liver lipids by MB07811 results from accelerated hepatic fatty acid oxidation, a known consequence of hepatic TR activation, as reflected by increased hepatic mitochondrial respiration rates, changes in hepatic gene expression, and increased plasma acyl-carnitine levels. Transaminase levels remained unchanged, or were reduced, and no evidence for liver fibrosis or other histological liver damage was observed after treatment with MB07811 for up to 10 weeks. Additionally, MB07811, unlike T(3), did not increase heart weight or decrease pituitary thyroid-stimulating hormone beta (TSHbeta) expression. CONCLUSION: MB07811 represents a novel class of liver-targeted TR agonists with beneficial low-density lipoprotein cholesterol-lowering properties that may provide additional therapeutic benefit to hyperlipidemic patients with concomitant NAFLD.

Relative solvation free energies calculated using an ab initio QM/MM-based free energy perturbation method: dependence of results on simulation length.

Molecular dynamics (MD) simulations in conjunction with thermodynamic perturbation approach was used to calculate relative solvation free energies of five pairs of small molecules, namely; (1) methanol to ethane, (2) acetone to acetamide, (3) phenol to benzene, (4) 1,1,1 trichloroethane to ethane, and (5) phenylalanine to isoleucine. Two studies were performed to evaluate the dependence of the convergence of these calculations on MD simulation length and starting configuration. In the first study, each transformation started from the same well-equilibrated configuration and the simulation length was varied from 230 to 2,540 ps. The results indicated that for transformations involving small structural changes, a simulation length of 860 ps is sufficient to obtain satisfactory convergence. In contrast, transformations involving relatively large structural changes, such as phenylalanine to isoleucine, require a significantly longer simulation length (>2,540 ps) to obtain satisfactory convergence. In the second study, the transformation was completed starting from three different configurations and using in each case 860 ps of MD simulation. The results from this study suggest that performing one long simulation may be better than averaging results from three different simulations using a shorter simulation length and three different starting configurations.

A Long-Acting PYY3-36 Analog Mediates Robust Anorectic Efficacy with Minimal Emesis in Nonhuman Primates.

The gut hormone PYY3-36 reduces food intake in humans and exhibits at least additive efficacy in combination with GLP-1. However, the utility of PYY analogs as anti-obesity agents has been severely limited by emesis and rapid proteolysis, a profile similarly observed with native PYY3-36 in obese rhesus macaques. Here, we found that antibody conjugation of a cyclized PYY3-36 analog achieved high NPY2R selectivity, unprecedented in vivo stability, and gradual infusion-like exposure. These properties permitted profound reduction of food intake when administered to macaques for 23 days without a single emetic event in any animal. Co-administration with the GLP-1 receptor agonist liraglutide for an additional 5 days further reduced food intake with only one animal experiencing a single bout of emesis. This antibody-conjugated PYY analog therefore may enable the long-sought potential of GLP-1/PYY-based combination treatment to achieve robust, well-tolerated weight reduction in obese patients.