Our in vivo models of hepatic steatosis with perturbed insulin concentrations had relatively increased IRS-1 expression at high insulin concentrations (T2FLD), and relatively increased IRS-2 at low insulin concentrations (T1FLD) (). This relative ‘imbalance’ between the two IRS molecules was associated with an up-regulation of FATP-2 & 5 in both states. Holding other parameters constant in vitro, we replicated this bimodal TG accumulation function simply by varying insulin levels. We thus describe a novel insulin driven bimodal FATP-lipid accumulation response.
FATP (later named FATP1) was first identified in 1994 by Schaffer et al and shown to increase the uptake of long chain fatty acids across the plasma membrane 
. The murine Fatp1
gene was found to span approximately 16 kilobases and contain 13 exons, of which exon 2 was shown to be alternatively spliced. Since then multiple groups have worked on various isoforms of FATP in different tissues of interest. Further, human relevance has been described and researched in the setting of X-linked adrenoleukodystrophy, a genetic neurodegenerative disorder wherein increased levels of saturated very long-chain fatty acids are found in tissues and plasma. Herein FATP2 has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase 
. Further, in adipocytes FATP1
has been shown to be transcriptionaly regulated by insulin 
. Thus, given the putative role of FATPs in hepatic lipid transport and their potential to be regulated by insulin in other tissues we set about looking into the link between insulin and lipid transport and its relation to FATP 2 and 5 in the liver.
Investigating the hyperinsulinemic state Kerouz et al
. have reported that obese ob/ob mice have significantly higher IRS-1 than IRS-2 liver protein 
. Furthermore, Guo et al
. reported that inactivation of IRS-1 leads to improvement in murine hepatic steatosis 
. Further, Taniguchi et al
. found that short term adenovirus mediated inactivation of IRS-2 increased hepatic steatosis 
. Thus, we conclude from the literature our data that an imbalance of IRS signaling favoring relatively more IRS-1 than IRS-2 occurs in the hyperinsulinemic state.
Investigating the hypoinsulinemic state Rojas et al
. observed that liver IRS-2 protein increased relative to IRS-1 after a 72-hour fast or with STZ-induced T1DM 
. Contrary to these findings, Simmgen et al
. reported that IRS-2 signaling is not required for hepatic lipid metabolism 
. However, the authors of these studies did not assess this in either fasting or STZ-induced conditions, where IRS-2 is shown to be increased. We observed in our insulin replacement experiment a dose dependent reduction in liver TG content in STZ treated T1FLD mice receiving insulin. Interestingly we also observed a significant decrease in fasting FFA plasma levels associated with increasing exogenous insulin doses. Though we did not measure fatty acid transport directly in our current experiments this is clearly an important path of future research. This decrease in FFA maybe a manifestation of direct insulin mediated reduction in peripheral lipolysis and therefore contribute to the reduction in TG accumulation independent of FATP regulation.
We acknowledge that these two in vivo models may not be completely translatable to human conditions such as type 2 and type 1 diabetes, however, further evidence that insulin impacts the FATP levels directly comes from our in vitro studies where targeted gene disruption of IRS-1&2, led to decreased FATP-2&5 expressions. Though all in vitro experiments were not performed under identical conditions (siRNA knockdown required an additional 12 hours of incubation, resulting in a different magnitude of TG accumulation) unlike in vivo, there is no peripheral lipolysis in vitro, thus we concluded that increased FATP expression at extremes of insulin concentrations is likely mediated via imbalanced IRS signaling. We acknowledge that some of the concentrations of insulin used (e.g. 100 mU/ml) maybe be supraphysiological but they do provide for proof of concept.
Indeed, others have also found that IRS-1 mRNA and protein levels are increased with insulin treatment 
, and that IRS-2 mRNA is down regulated by insulin 
. Thus, if a relative increase of IRS-1 signaling is paramount in the pathogenesis of obesity comorbidities, then a pharmacologic means of restoring IRS-2 signaling might prove to be a viable therapeutic option. White et al
have reported that finding drugs which stimulate IRS-2 synthesis or promote its signaling might be a useful treatment option for obesity-associated T2DM 
. Similarly, Gupta et al.
have reported that the long-acting glucagon-like peptide 1 (GLP-1) agonist, Exendin-4, decreases hepatic steatosis and activates the same pathway as IRS-2 
. Also of note, insulin has been shown in other tissues, such as cardiac myocytes and adipocytes, to increase fatty acid uptake 
. Furthermore, drugs used to treat T2DM, which also improve hepatic steatosis, such as rosiglitazone 
and metformin 
, are found to preferentially increase and restore IRS-2 expression.
The main finding of this report is that the amount of circulating insulin is a major modulator of hepatic steatosis via regulation of liver fatty acid transport proteins. In both in vitro and in vivo experiments, insulin-mediated TG accumulation in the liver exhibited a bimodal function, where both hypo and hyperinsulinemia led to augmented liver fat storage. While we observed reduced FATP 2 and 5 in these experiments, the reduction in TG levels could also be attributable to enhanced fatty acid oxidation, decreased de novo lipogenesis or altered lipoprotein export. These pathways definitely need to be evaluated in future experiments in order to understand the contribution of altered FFA uptake. Ultimately, the sums of all of these processes appear to be mediated via an imbalance of insulin substrates, where hypoinsulinemia is characterized by excessive IRS-2 signaling, and hyperinsulinemia with predominant IRS-1 signaling. Regardless as to which side the equilibrium is shifted; imbalanced insulin signaling points to a common potential FATP response. The identification and verification of this novel link in follow up experiments may provide future therapeutic targets for the treatment of obesity associated fatty liver disease.