Lost in Translation

There are few methodologies that have done more to advance our understanding of insulin action in humans and animals than the hyperinsulinemiceuglycemic clamp (henceforth referred to as the insulin clamp or clamp). The clamp was first developed for use in humans by DeFronzo, Tobin, and Andres at Johns Hopkins in 1979 (1). This technique was transferred to the rat in 1983 (2,3) and the mouse in the early 1990s (4,5). The insulin clamp is often combined with isotopic methods so that the investigator cannot only determine a total wholebody insulin action but also how specific tissues and metabolic pathways are affected. The adaptation of the insulin clamp for use in the mouse has been critical to the characterization of the still growing number of mouse models with modifications to the genes involved in the regulation of glucose and energy homeostasis. Despite the importance of this tool, a significant deficiency has evolved in the process of translating the clamp from humans to mice. The first articles reporting insulin clamps in the mouse conscientiously presented methods and results (4,5). However, since these first studies in the mouse, the care and conventions of the human clamp literature that immediately followed its development have largely been traded for no, minimal, or inaccurate description of methods and inadequate presentation of results. The reader is provided with too little information to independently interpret the results of an article. Instead, the reader is solely dependent on the conclusion to which he or she is directed by the authors. The perception that standards in the human and rat literature have lowered in recent years was brought to our attention during the preparation of this article. Here, we focus on the mouse literature. Of course, sound reporting of clamp methods and results applies to all species. The purpose of this article is to see how the careful presentations of the initial human insulin clamp studies might be better translated to the mouse insulin clamp literature. We will outline the problem and cite specific examples where failure to present clamps in a complete and accurate manner has had a negative impact. We will convey our view as to what investigators using the insulin clamp should include in published reports and what reviewers should require of them so that the reader can independently interpret the experiment. Vagaries. The insulin clamp is challenging and subject to variations that are specific to the laboratory in which they are conducted. Despite these challenges and nuances, the clamp has been the object of very little scrutiny when applied to the mouse. Ayala et al. (6) first reported this deficit in 2006. Our sense is that some improvements have occurred with regards to the description of insulin clamps since this first statement of the problem. Nevertheless, many problems remain. Let us provide some examples. In 2004 and 2005, nearly half (44%) of studies in mice failed to report a sampling site (6). In the 2 years following the publication of Ayala et al. (6), this number has dropped to 30%. Although this is an improvement, failure to indicate a sampling site in nearly one-third of published studies remains a deficiency in the field. One area where little has changed in the mouse clamp literature is the reporting of insulin doses and insulin levels. Prior to 2006, 48% of studies in mice mention a priming dose of insulin but fail to indicate the actual dose. For reasons described in detail later, this is not a trivial point. Furthermore, in almost all of these studies, absolute insulin levels (fasting and clamp) were not reported. It is impossible for a reader to know that hyperinsulinemia has been achieved and insulin levels are comparable between groups if insulin levels are not reported. Since 2006, the percentage of published reports that fail to indicate the priming dose of insulin and insulin levels was still 40%. Finally, to date 90% of reports where insulin clamps were performed in mice do not show a time course of glucose levels and glucose infusion rates. Without this information, one cannot know whether a steady-state clamp has been achieved. Nearly all clamp experiments in humans present these three parameters: sampling site, insulin dosing/levels, or time course of glucose and glucose infusion. It is therefore unclear to us why standards for the mouse clamp literature should be any different. The previous paragraph describes some fundamental omissions in the mouse clamp literature. Without the presentation of fundamental clamp components such as time course of glucose and glucose infusion it is impossible to know whether a clamp has been performed. This is not to say that from time to time certain vital information may be inadvertently left on the cutting room floor. A minimalist approach to the description of procedure and presentation of results is almost convention. A curious tangential observation is that whereas insulin clamps in the mouse are often poorly described or not described at all, glucose tolerance tests and insulin tolerance tests are described reasonably well. Methodology is usually adequately presented, and time courses are shown. The results from well-conducted glucose tolerance tests and insulin tolerance tests are more interpretable than an insulin clamp that does not report clamp insulin concentrations. In the same vein, there are articles that provide little to no information on the clamp but will describe a routine published buffer system or assay with great detail. The question thus remains, why has the insulin clamp been lost in translation to the mouse? An example illustrating the vagaries that are pervasive in the mouse clamp literature is evident in the ambiguous From the Department of Molecular Physiology and Biophysics, Vanderbilt Mouse Metabolic Phenotyping Center, Vanderbilt University School of Medicine, Nashville, Tennessee. Corresponding author: David H. Wasserman, david.wasserman@vanderbilt.edu. Received 11 April 2009 and accepted 10 June 2009. DOI: 10.2337/db09-0537 © 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. PERSPECTIVES IN DIABETES