The role of intramuscular or intramyocellular lipids (IMCLs) in insulin resistance has been extensively investigated over the past 30 years employing model systems and human tissue analyses. This line of inquiry has evolved from the early hypothesis that intramuscular triglycerides (IMTGs) cause insulin resistance to the more recent heated debate over which specific lipids within muscle are the true lipotoxic culprits. This progression in knowledge does not dissuade from the position put forth herein that excess IMCLs cause insulin resistance. First, the case needs a framework. Let us start with some inarguable truths. Insulin resistance is part of a syndrome influenced by obesity, physical activity, nutrition and other lifestyle factors, and likely genetics. Though the exact causes of insulin resistance remain elusive, various perturbations in diet, lipid oversupply and exercise have been demonstrated experimentally and observationally to alter both IMCLs and insulin resistance. The following background and subset of key studies provide important physiological context and a mechanistic basis supporting a causal role of IMCLs in insulin resistance. Fatty acids have long been known to serve as an important fuel source for exercising muscle (Himwich & Rose, 1929). IMTGs were first described in rat muscle by Denton & Randle (1967), and soon thereafter were first reported to be utilized during exercise (Carlson et al. 1971). Guided by early studies by Randle et al. who reported a reciprocal regulation of glucose and fatty acid metabolism (Randle et al. 1963), several lines of investigation concluded that reduced oxidative capacity for fatty acid oxidation within skeletal muscle played a role in insulin resistance (McGarry, 1992; Kelley et al. 1999; Bruce et al. 2003) and type 2 diabetes (Kelley & Simoneau, 1994). Thus, while healthy muscle can effectively use a variety of fuel sources, including IMTGs, insulin resistance and type 2 diabetes are caused by an imbalance of nutrient supply and utilization. These reports linking a reduced capacity for fatty acid oxidation with insulin resistance were consistent with early studies across animal models (Storlien et al. 1991) and in humans (Phillips et al. 1996; Pan et al. 1997; Krssak et al. 1999; Perseghin et al. 1999; Goodpaster et al. 2000a,b) correlating IMTG content with insulin resistance and type 2 diabetes. There remained, however, unresolved questions about positive and negative roles for IMTGs in health and disease. This quandary was highlighted by the ‘athlete's paradox’ (Goodpaster et al. 2001), in which endurance trained athletes have similar IMTG content to individuals with type 2 diabetes, yet are very insulin sensitive. This study, supported by other exercise training studies (Dube et al. 2008, 2011; Coen et al. 2015) in humans and more mechanistic studies in model systems (Itani et al. 2002; Yu et al. 2002) demonstrating that IMTGs were quite likely not the true cause of insulin resistance, helped to move the field forward towards a deeper more sophisticated understanding of the role for other lipid intermediates in insulin resistance. Over the past two decades, numerous subsequent studies have attempted to define ‘good’ and ‘bad’ muscle lipids and to disentangle the conundrum of why IMTGs are not consistently associated with insulin resistance. Data from both model systems and human muscle biopsy studies have revealed, with the aid of more advanced mass spectrometry, that specific IMCL species, primarily diacylglycerol (DAG) and sphingolipids, are more likely to cause insulin resistance than are IMTGs (Bergman et al. 2009, 2012, 2016; Coen et al. 2010, 2015; Dube et al. 2011; van Hees et al. 2011). In a 2016 CrossTalk article, Summers and Goodpaster put forth the position that ceramides, part of the larger sphingolipid family, cause insulin resistance (Summers & Goodpaster, 2016). Ceramides and their precursor dihydroceramides are converted into complex sphingolipids within the Golgi apparatus, or are deacylated by ceramidases within other cellular compartments, e.g. lysosomes. Pharmaceutical or genetic interventions to slow ceramide synthesis or accelerate its degradation consistently enhance insulin sensitivity in rodent models (Holland et al. 2007; Yang et al. 2009; Ussher et al. 2010; Li et al. 2011; Dekker et al. 2013) and prevent lipid-induced insulin resistance. Inhibition of ceramide synthesis (i.e. myriocin, fumonisin B1, or siRNA-mediated knockdown of Spt subunits or Des-1) or stimulation of ceramide degradation (e.g. acid ceramidase overexpression) negates lipid-antagonism of insulin signalling in these systems (Chaurasia & Summers, 2015). The majority of data obtained from IMCL profiling and quantification in human muscle biopsies support strong associations between muscle ceramides and insulin resistance (Adams et al. 2004; Straczkowski et al. 2004; Coen et al. 2010). Insulin-sensitizing weight loss and exercise interventions consistently decrease ceramides (Dube et al. 2008, 2011; Coen et al. 2015). Human studies have also revealed associations between circulating sphingolipids and insulin resistance (Haus et al. 2009), which is consistent with an emerging hypothesis that ceramides may enter the muscle from the circulation (Boon et al. 2013). Moreover, the mechanisms of insulin sensitization with adiponectin and fibroblast growth factor 21 appear to implicate tissue ceramide reduction (Xia et al. 2015). DAG is a lipid intermediate in IMTG synthesis and lipolysis as well as an intracellular second messenger; when in excess, it is capable of increasing the activity of protein kinase C (PKC) (Spitzer et al. 1989). Activated PKCs reduce insulin-stimulated glucose transporter type 4 translocation and glucose uptake through serine phosphorylation of insulin receptor substrate 1, inhibiting kinase activity and inactivating phosphoinositide 3-kinase and protein kinase B/Akt (Szendroedi et al. 2014). The data are mixed, however, on a role for the total amount of DAG in muscle insulin resistance. This could possibly be explained by the lack of consistency across lipidomics platforms to accurately quantify the most relevant molecular species. Indeed, emerging evidence suggests that the specific molecular species of DAG and their subcellular location may produce their biological effects in this regard. Enzymatic synthesis, elongation and desaturation, along with variation in dietary fatty acid intake, lead to a multitude of IMCL species of varying fatty acid chain length and stereoisomer specificity. More than 30 years ago 1,2-DAG isomers were shown to uniquely activate PKC (Rando & Young, 1984). Aided by advances in mass spectrometry, specific DAG (Amati et al. 2011; Bergman et al. 2012) and ceramides (Chung et al. 2017; Perreault et al. 2018), but not the total amount of IMCL within these general classes, have been reported to be implicated in insulin resistance. This is highlighted by a recent study by Chaurasia et al. (2019) who provide compelling mechanistic evidence that specific ceramide species cause insulin resistance. Emerging evidence shows that localization of triglycerides, diacylglycerols and sphingolipids appears to play an important role in promoting decreased insulin sensitivity (Jocken et al. 2013; Szendroedi et al. 2014; Chung et al. 2017; Perreault et al. 2018). Recent studies from the Bergman laboratory indicate that the content and distribution of DAG isomers and species in sarcolemmal, cytosolic, nuclear and mitochondrial/endoplasmic reticulum compartments provide specific signatures of skeletal muscle insulin resistance (Perreault et al. 2018). Ceramides and other sphingolipids are also known to be distributed within various cellular compartments. Recent evidence indicates that excess ceramides which are not localized within the cytosol, particularly saturated ceramide species, are features of insulin-resistant muscle (Chung et al. 2017; Perreault et al. 2018). Taken together, these recent data on the specific molecular species and location of IMCLs within muscle have further supported a causal role for IMCLs in insulin resistance. During the past 30 years of prosecuting IMCLs as a malefactor in insulin resistance, the evidence implicating a causal role for these complex lipids has become clearer. Only specific molecular species of IMCLs within particular cellular compartments cause insulin resistance. There is, however, still much to learn. Further understanding of the particular molecular IMCL species and their subcellular location will be crucial to understand how altering these specific lipids and their molecular pathways can impact metabolic function and insulin sensitivity. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a ‘Last Word’. Please email your comment, including a title and a declaration of interest, to [email protected]. Comments will be moderated and accepted comments will be published online only as ‘supporting information’ to the original debate articles once discussion has closed. Bret Goodpaster, PhD is the Scientific Director at the AdventHealth Translational Research Institute (TRI). He has published over 250 peer-reviewed papers, review articles and book chapters. He has served on Editorial Boards for Diabetes, the American Journal of Physiology, the Journals of Gerontology, and is currently an Associate Editor for Diabetologia. He obtained a BS in Biology from Purdue, and after completing a Pre-doctoral Fellowship at Maastricht University in the Netherlands, received his PhD in Human Bioenergetics from Ball State University in 1995. Prior to coming to the AdventHealth TRI, he was Professor of Medicine at the University of Pittsburgh. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None. Sole author. None. The author thanks the many colleagues and collaborators over the years who have contributed to these studies and this debate.