The objective of this study was to employ a novel computational platform to gain mechanistic insight into the molecular changes induced by pharmacological inhibition of DGAT1. Acute gene expression changes were utilized to infer multiple overlapping molecular regulators of lipid and carbohydrate metabolism predictive of benefits of DGAT1 inhibition such as lipid lowering and improved insulin sensitivity. Our analysis allows us to postulate the molecular network conferring these metabolic benefits to better understand the mechanism of action for pharmacological inhibition of DGAT1.
Our understanding of the physiologic role of DGAT1 stems largely from studies of genetically modified mice that lack DGAT1 from birth. It is noteworthy that this analysis focused on transcriptomics in the jejunum elicited by the administration of a pharmacological inhibitor of DGAT1 in an adult rat which suggests similar molecular phenotype to DGAT1 knockout mice. Recently, DGAT1 knockout mice were shown to have decreased expression of PPARalpha, gamma and delta as well as target genes suggestive of reduced lipid uptake and metabolism and increase glucose uptake 
which is consistent with our top ranking hypotheses. Additionally, DGAT-1 deficient mice demonstrate resistance to weight gain on high fat diet, improved insulin sensitivity and a lower percentage of oleic acid in their skeletal muscle and adipose tissue triglyceride 
. Again, our CRE generated hypotheses identified reversal of high fat diet, reduced insulin resistance and decreased oleic acid. These data support the notion that the intestine is an important tissue involved in whole body insulin sensitivity diet-induced obesity. Insulin resistance in the intestine has been associated with increased apolipoproteins, chylomicrons, de novo lipogenesis, and increased fatty acid and cholesterol uptake via CD36 and SCARB1 
. In our study not only was triglyceride synthesis decreased via inhibition of the target, but transcription of the key apolipoproteins for chylomicron synthesis (ApoB, ApoA I, ApoA IV, and ApoC III) were reduced. Of these Apo CIII was the most dramatic (see Table S1
) with greater that a 5 fold reduced expression at the high dose. The expression and secretion of ApoC III is increased in insulin resistant states and plasma circulating levels are higher in metabolic syndrome and type II diabetes 
. Finally, Lee et al demonstrated that intestine specific expression of DGAT1 in the DGAT1 deficient mice prevented the knockout mouse from being resistant to diet induced obesity 
In contrast, DGAT1 knockout mice are hyperphagic 
; whereas, administration of PF-04620110 results in a decrease in food intake. Our working hypothesis is that elevated levels of incretin hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are at least in part mediating this response (). It is our belief that decreased food intake is an integral part of the mechanism of action driving a metabolically favorable profile following pharmacological inhibition of DGAT1 and thereby did not try to dissociate food intake dependent effects from food intake independent effect in our analysis. Normal lipid absorption entails the breakdown of dietary triglyceride into free fatty acids and 2-monoacylglycerol by pancreatic lipases in the lumen of the small intestine. This allows transport of the free fatty acids into the enterocytes where they can be re-esterified and packaged into chylomicrons for delivery to the circulation. Clearly the major role of DGAT1 in triglyceride synthesis and intestinal lipid absorption has been demonstrated 
with DGAT1 accounting for 89% of triglyceride synthesis in rat intestinal membranes. Theoretically, DGAT1 inhibition would cause an immediate build up of its substrates, diacylglycerol and free fatty acids. Polyunsaturated fatty acids have been demonstrated to decrease the expression of lipogenic genes via SREBP promoter elements (SRE) 
. Therefore DGAT1 inhibition would result in decreased lipogenesis in the intestine driven by an excess of free fatty acids. There has been mounting evidence in high fat diet rodent models and humans supporting a negative impact of de novo lipogenesis and monounsaturated fatty acid synthesis on insulin sensitivity 
. Mice fed high fat western diet for one week demonstrate a robust increase in the expression of intestinal SREBF1 and SCD-1, and develop insulin resistance with little change in hepatic gene expression 
. Coincidentally, SREBF1 and SCD1 where robustly down regulated in the jejunum but unchanged in the liver with DGAT1 inhibition. Furthermore CRE hypotheses for reduced SREBF1, PPARa, RXR, MLX, and PGC1a all suggest a decrease in fatty acid synthesis, while the decrease in SCD1 may be contributing to the depletion of oleic acid, and secondary enrichment in arachidonic acid (). Recent evidence has indicated a benefit for a high ratio of C20-C22 PUFAS to saturated and monounsaturated fatty acids for improved glycemic control and insulin sensitivity 
. Thus an additional effect of DGAT1 inhibition would be the insulin sensitizing effect of enriched very long chain PUFA.
The Causal Reasoning approach has the advantage of providing detailed molecular hypotheses on potential causal drivers of observed expression changes. Each assertion can be followed back to the primary literature providing confidence to the researcher to follow-up on the computational predictions. In some cases the predicted direction of the CRE hypothesis may conflict with the observed direction of the transcript change. For example, a CRE hypothesis of decreased CFTR
protein and/or activity conflicts with the observed increase in transcripts for CFTR as well as Annexin 2 and S100A10 that complex with CFTR enabling its function 
. The literature evidence supporting the CFTR
hypothesis came from two studies in CFTR knockout mice 
. Regulated genes in this context may include compensatory and/or regulatory feedback gene expression changes which in turn may complicate the interpretation of some of the CRE hypotheses. One possibility is that a CRE hypothesis may represent protein level or activity which is not necessarily reflective of the mRNA level or that the CRE hypothesis is based on gene changes in response to an initial decrease in CFTR protein or activity that led to feedback increase in transcript level hence reflecting an earlier temporal event. Another example is the CRE hypotheses for increased fatty acid oxidation enzymes Enoyl-CoA hydratase (EHHADH), and hydroxysteroid (17-beta) dehydrogenase 4 (HSD17B4). Both of these hypotheses are supported by the same transcript evidence from a single literature source 
. Moreover, the same transcript evidence is completely subsumed under the much higher ranking hypothesis of decreased PPAR alpha, which includes decreased transcription of EHHADH and HSD17B4 which could be an effect of a feedback loop.
Clearly, the hypotheses as well as the resulting model can only be as good as the underlying causal relationships. Consequently, the method is unlikely to uncover completely novel areas of biology. However, it can provide novel insights by reporting upstream drivers to be relevant in a certain context. As efforts to curate larger parts of the biomedical literature are underway, we expect the power of the approach to increase.
We have employed the causal reasoning approach as a means of visualizing an extensive and diverse set of gene expression changes to generate high level molecular hypotheses that will enable a better understanding of the anti-adipogenic and anti-diabetic benefits derived following pharmacological inhibition of DGAT1. Additionally, this analysis has allowed us to understand the advantages and limitations of causal reasoning. The approach has allowed us to confirm in a systematic fashion that pharmacological inhibition of DGAT1 in adult rats generates molecular hypotheses that are consistent with the metabolically beneficial phenotype of mice lacking DGAT1.