CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

Mechanism of glucose intolerance in mice with dominant negative mutation of CEACAM1

Mice with liver-specific overexpression of dominant negative phosphorylation-defective S503A-CEACAM1 mutant (L-SACC1) developed chronic hyperinsulinemia resulting from blunted hepatic clearance of insulin, visceral obesity, and glucose intolerance. To determine the underlying mechanism of altered glucose homeostasis, a 2-h hyperinsulinemic euglycemic clamp was performed, and tissue-specific glucose and lipid metabolism was assessed in awake L-SACC1 and wild-type mice. Inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) caused insulin resistance in liver that was mostly due to increased expression of fatty acid synthase and lipid metabolism, resulting in elevated intrahepatic levels of triglyceride and long-chain acyl-CoAs. Whole body insulin resistance in the L-SACC1 mice was further attributed to defects in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Insulin resistance in peripheral tissues was associated with significantly elevated intramuscular fat contents that may be secondary to increased whole body adiposity (assessed by (1)H-MRS) in the L-SACC1 mice. Overall, these results demonstrate that L-SACC1 is a mouse model in which chronic hyperinsulinemia acts as a cause, and not a consequence, of insulin resistance. Our findings further indicate the important role of CEACAM1 and hepatic insulin clearance in the pathogenesis of obesity and insulin resistance.     

Th2 Cytokines Inhibit Lymphangiogenesis

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.     

Clinical considerations for the development of biosimilars in oncology

Despite availability of biologic therapies, limited patient access to many of the most-effective cancer treatments affects overall health outcomes. To address this issue, many governments have enacted legislation for the approval of biosimilars. The term “biosimilar” refers to a biologic product that is developed to be highly similar, as opposed to identical, to a licensed biologic product (the reference or innovator product), such that, per US Food and Drug administration draft guidelines, “no clinically meaningful differences [exist] between the biological product and the reference product in terms of safety, purity, and potency.” This article presents some considerations about the development of biosimilars in cancer treatment through an overview of biosimilars from a clinical perspective. Topics covered include the development requirements and unique regulatory requirements for biosimilars, labeling considerations, potential limitations to the uptake of biosimilars, and review of some biosimilars in development for oncology indications.     

Suppression of Adaptive Immune Cell Activation Does Not Alter Innate Immune Adipose Inflammation or Insulin Resistance in Obesity

Obesity-induced inflammation in visceral adipose tissue (VAT) is a major contributor to insulin resistance and type 2 diabetes. Whereas innate immune cells, notably macrophages, contribute to visceral adipose tissue (VAT) inflammation and insulin resistance, the role of adaptive immunity is less well defined. To address this critical gap, we used a model in which endogenous activation of T cells was suppressed in obese mice by blocking MyD88-mediated maturation of CD11c⁺ antigen-presenting cells. VAT CD11c⁺ cells from Cd11cCre ⁺ Myd88 ^fl/fl vs. control Myd88 ^fl/fl mice were defective in activating T cells in vitro, and VAT T and B cell activation was markedly reduced in Cd11cCre ⁺ Myd88 ^fl/fl obese mice. However, neither macrophage-mediated VAT inflammation nor systemic inflammation were altered in Cd11cCre ⁺ Myd88 ^fl/fl mice, thereby enabling a focused analysis on adaptive immunity. Unexpectedly, fasting blood glucose, plasma insulin, and the glucose response to glucose and insulin were completely unaltered in Cd11cCre ⁺ Myd88 ^fl/fl vs. control obese mice. Thus, CD11c⁺ cells activate VAT T and B cells in obese mice, but suppression of this process does not have a discernible effect on macrophage-mediated VAT inflammation or systemic glucose homeostasis.     

BIOCHEMICAL GENETICS OF FUNDULUS HETEROCLITUS (L.). VI. GEOGRAPHICAL VARIATION IN THE GENE FREQUENCIES OF 15 LOCI

Geographic variation in the gene frequencies corresponding to 15 polymorphic enzymes were studied in the common killifish Fundulus heteroclitus. Aat-A, Est-B, Fum-A, H6pdh-A, Mpi-A and Pgm-B showed clinal variation in allelic frequencies along the Atlantic coast of North America, while Aat-B, Ap-A, and the EST-C phenotypes did not. The clinal allelic variation of six previously examined loci (Ldh-B, Mdh-A, Gpi-B, Idh-A, Pgm-A, and 6-Pgdh-A) was extended to locations farther north. Gene diversity was lowest in the cold waters of northern latitudes and highest in the warmer waters of southern latitudes. The variety of clinal shapes and widths suggests that selection has affected the allelic distributions for at least some of these loci. This hypothesis is discussed with regard to the range contractions and extensions caused by the glacial advances and retreats during the Pleistocene.     

The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor

BMS-707035 is an HIV-1 integrase strand transfer inhibitor (INSTI) discovered by systematic optimization of N-methylpyrimidinone carboxamides guided by structure-activity relationships (SARs) and the single crystal X-ray structure of compound 10. It was rationalized that the unexpectedly advantageous profiles of N-methylpyrimidinone carboxamides with a saturated C2-substitutent may be due, in part, to the geometric relationship between the C2-substituent and the pyrimidinone core. The single crystal X-ray structure of 10 provided support for this reasoning and guided the design of a spirocyclic series 12 which led to discovery of the morpholino-fused pyrimidinone series 13. Several carboxamides derived from this bicyclic scaffold displayed improved antiviral activity and pharmacokinetic profiles when compared with corresponding spirocyclic analogs. Based on the excellent antiviral activity, preclinical profiles and acceptable in vitro and in vivo toxicity profiles, 13a (BMS-707035) was selected for advancement into phase I clinical trials.     

Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I(P)) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I(P) because of the following: (1) it was absent in 0 mM [K(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.     

The SNAP-25 linker as an adaptation toward fast exocytosis

The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways.     

Regulation of alternative oxidase gene expression in soybean

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 °C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.