Among the most widely used animal models in obesity-induced type 2 diabetes mellitus (T2DM) research are the congenital leptin- and leptin receptor-deficient rodent models. These include the leptin-deficient ob/ob mice and the leptin receptor-deficient db/db mice, Zucker fatty rats, Zucker diabetic fatty rats, SHR/N-cp rats, and JCR:LA-cp rats. After decades of mechanistic and therapeutic research schemes with these animal models, many species differences have been uncovered, but researchers continue to overlook these differences, leading to untranslatable research. The purpose of this review is to analyze and comprehensively recapitulate the most common leptin/leptin receptor-based animal models with respect to their relevance and translatability to human T2DM. Our analysis revealed that, although these rodents develop obesity due to hyperphagia caused by abnormal leptin/leptin receptor signaling with the subsequent appearance of T2DM-like manifestations, these are in fact secondary to genetic mutations that do not reflect disease etiology in humans, for whom leptin or leptin receptor deficiency is not an important contributor to T2DM. A detailed comparison of the roles of genetic susceptibility, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and diabetic complications as well as leptin expression, signaling, and other factors that confound translation are presented here. There are substantial differences between these animal models and human T2DM that limit reliable, reproducible, and translatable insight into human T2DM. Therefore, it is imperative that researchers recognize and acknowledge the limitations of the leptin/leptin receptor-based rodent models and invest in research methods that would be directly and reliably applicable to humans in order to advance T2DM management.

Obesity and type 2 diabetes mellitus (T2DM) are rapidly growing worldwide epidemics with major health consequences. Various human-based studies have confirmed that both genetic and environmental factors (particularly high-caloric diets and sedentary lifestyle) greatly contribute to human T2DM. Interactions between obesity, insulin resistance and β-cell dysfunction result in human T2DM, but the mechanisms regulating the interplay among these impairments remain unclear. Rodent models of high-fat diet (HFD)-induced obesity have been used widely to study human obesity and T2DM. With >9000 publications on PubMed over the past decade alone, many aspects of rodent T2DM have been elucidated; however, correlation to human obesity/diabetes remains poor. This review investigates the reasons for this translational discrepancy by critically evaluating rodent HFD models. Dietary modification in rodents appears to have limited translatable benefit for understanding and treating human obesity and diabetes due—at least in part—to divergent dietary compositions, species/strain and gender variability, inconsistent disease penetrance, severity and duration and lack of resemblance to human obesogenic pathophysiology. Therefore future research efforts dedicated to acquiring translationally relevant data—specifically human data, rather than findings based on rodent studies—would accelerate our understanding of disease mechanisms and development of therapeutics for human obesity/T2DM.

Summaryonly a limited number of anti-diabetic drugs are in clinical use for humans, most of which have adverse health effects, but little impact on disease progression, and none of which cures t2DM or clearly prolongs life.the purpose of this review is to examine the underlying molecular, biological, and physiological differences -from gene regulation to whole-animal and population levels -that help explain why rodents do not serve as reliable models for studying human t2DM. this review will also address how researchers may overcome this translational barrier by employing a wide range of human-based investigational methods that will promote human-relevant discoveries while reducing -and eventually replacing -the use of animals in t2DM research.

The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A(1)AR, A(2a)AR, A(2b)AR, and A(3)AR. The A(1)AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A(2a)AR and A(2b)AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A(2a)AR knockout (KO), and A(2b)AR KO mice. The A(2a)AR agonist CGS-21680 did not alter basal contractility or β-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A(2a)AR KO hearts. However, A(2a)AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A(1)AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective A(2b)AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A(1)AR antiadrenergic effect was not potentiated in A(2b)AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A(2a)AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A(1)AR antiadrenergic effect, whereas A(2b)AR exerts direct contractile effects but does not alter β-adrenergic or A(1)AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function.

Chandrasekera PC, Kargacin ME, Deans JP, Lytton J. Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3. Am J Physiol Cell Physiol 296: C1105-C1114, 2009. First published February 18, 2009 doi:10.1152/ajpcell.00650.2008.-The sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca 2ϩ concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca 2ϩ ; however, Ca 2ϩ affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca 2ϩ uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca 2ϩ activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/ IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca 2ϩ affinity for SERCA3 compared with SERCA2b (1.10 Ϯ 0.04 vs. 0.26 Ϯ 0.01 M), and using mixtures of recombinant protein isoforms, we validated the twocomponent uptake model. Then we determined apparent Ca 2ϩ affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca 2ϩ affinity in these two preparations was 1.04 Ϯ 0.07 and 1. ]), as well as the levels of Ca 2ϩ in the endoplasmic reticulum stores required for controlling complex dynamics of Ca 2ϩ signals. SERCA proteins are encoded by a family of structurally related and alternatively spliced transcripts from three distinct genes (SERCA1, SERCA2, and SERCA3) expressed in a tissue-dependent and/or developmentally regulated manner. Alternative splicing of the ATP2A1 gene generates transcripts encoding SERCA1a and SERCA1b isoforms, which are expressed exclusively in adult and neonatal fast-twitch skeletal muscle, respectively. The ATP2A2 gene encoding the SERCA2 protein is alternatively spliced and processed in a tissue-specific manner, giving rise to three isoforms: SERCA2a, which is expressed predominantly in cardiac/slow-twitch skeletal muscle; SERCA2b, which is expressed ubiquitously; and SERCA2c, which has been reported to have a broader expression pattern than that typically attributed to SERCA2a. The level of SERCA2c protein relative to coexpressed SERCA2a or SERCA2b is not known (13,19,35). SERCA2b has a higher apparent affinity for Ca 2ϩ and turns over at a rate twofold slower than SERCA2a. SERCA2c has a lower apparent Ca 2ϩ affinity and a slower turnover rate than SERCA2a. The functional differences are due to replacement of the COOH-terminal sequence AILE (amino acids 994 -997) in SERCA...

Much of Alzheimer disease (AD) research has been traditionally based on the use of animals, which have been extensively applied in an effort to both improve our understanding of the pathophysiological mechanisms of the disease and to test novel therapeutic approaches. However, decades of such research have not effectively translated into substantial therapeutic success for human patients. Here we critically discuss these issues in order to determine how existing human-based methods can be applied to study AD pathology and develop novel therapeutics. These methods, which include patient-derived cells, computational analysis and models, together with large-scale epidemiological studies represent novel and exciting tools to enhance and forward AD research. In particular, these methods are helping advance AD research by contributing multifactorial and multidimensional perspectives, especially considering the crucial role played by lifestyle risk factors in the determination of AD risk. In addition to research techniques, we also consider related pitfalls and flaws in the current research funding system. Conversely, we identify encouraging new trends in research and government policy. In light of these new research directions, we provide recommendations regarding prioritization of research funding. The goal of this document is to stimulate scientific and public discussion on the need to explore new avenues in AD research, considering outcome and ethics as core principles to reliably judge traditional research efforts and eventually undertake new research strategies.

dossier to the European Chemicals Agency (ECHA) containing an extensive list of data on the intrinsic properties of a substance, ranging from chemical characterization and physicochemical properties to toxicological and ecotoxicological data, with increasing demands depending on the tonnage band of the quantity of the substance placed on the market. In addition, registrants should collect information on use and exposure to perform a chemical safety assessment (CSA) based on the toxicity profile of the substance. The minimum data requirements are described in Annexes VI-X of the regulation. For toxicological testing, the standard require-

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported.