NAFLD is an underrecognized and increasingly prevalent condition that remains poorly understood. The recent publications showing FL in association with liver-specific deletion of GHR and STAT5 have focused attention on GH signaling and its role in the development of NAFLD and associated conditions (14
). Indeed, GH has long been implicated in the development of liver steatosis, yet the precise role of GH remains confusing, with reports that administration of GH both causes and cures FL (31
). Furthermore, no published mouse models of GH excess or deficiency have described FL (24
). Finally, while liver-specific disruption of GH signaling has now been shown to cause FL, surprisingly, FL has not been described in any mouse models with global disruption of GHR or STAT5 or in mice with liver-specific deletion of IGF-1 (24
We set out to determine the effect of liver-specific deletion of JAK2 in mice and were not at all surprised to observe that these JAK2L mice developed early and severe FL. In fact, the degree of fat accumulation in the livers of JAK2L mice was significantly greater than that observed in the liver-specific deletion of either GHR or STAT5, and the degree of steatosis was among the most severe of all genetic models of NAFLD in the published literature. The 20-week-old cohort of animals showed moderate to severe hepatocellular lipidosis, with mild accompanying foci of lobular inflammation and perisinusoidal fibrosis. These lesions resembled morphologically those seen in NASH, though without all of the features required or consistent with the diagnosis of NASH in humans. This shows that simple hepatic steatosis observed in younger mice progresses to lesions resembling mild NASH.
There were some differences between the JAK2L mice and the prior reports of the liver-specific deletions of GHR (GHRLD) and STAT5. Most notably, GHRLD mice were insulin resistant and glucose intolerant, while the JAK2L mice had normal glucose and insulin homeostasis (30
). This difference was unexpected and remains unexplained. One could speculate that these and other differences in phenotype relate to the fact that loss of JAK2 in hepatocytes is more complicated than simply disrupting GH signaling, with known effects on many other signaling pathways such as leptin and IL-6 (38
). One other notable difference was that expression of Igfbp3 was not significantly lower in JAK2L mice, while it was modestly reduced in the hepatocyte-specific deletion of STAT5 and was dramatically reduced in the GHRLD mice. However, despite the differences, the fundamental issues were the same. Namely, there was a significant reduction in expression of Igf1
in the liver, a concomitant near-total reduction in serum IGF-1, and a dramatic increase in serum GH. We observed other consequences of GH excess, including a degree of lipodystrophy with a decrease in total body fat and a rise in levels of plasma FFA. The effects on lipolysis were very similar to those described in humans with acromegaly or in transgenic mice with overexpression of GH (33
). Further, increased visceral adiposity was reported in mice with global disruption of STAT5, and a recent publication described increased body fat in mice with global disruption of GHR (44
). We reasoned that the development of FL in the liver-specific deletion of STAT5, GHR, and JAK2 were all at least somewhat related to dysregulated GH secretion and concomitant effects of increased GH on lipolysis. To test the effect of excessive GH secretion on development of FL, we crossed the JAK2L mice with the GH-deficient little
mice, and indeed, we found that development of FL was completely rescued in mice unable to augment GH secretion in the absence of serum IGF-1.
This striking observation demonstrated that the development of FL in all 3 models of liver-specific disruption of GH signaling was due, in part, to the loss of IGF-1–mediated feedback inhibition of GH secretion and the associated effects of dysregulated GH secretion on lipolysis. While excess GH secretion was necessary for development of FL in the JAK2L mice, it was also clearly not sufficient. Indeed, there are now 5 distinct classes of mouse models with excess plasma GH or GH action: (a) mice with direct overexpression of Gh
); (b) mice with global disruption in GH signaling (23
); (c) mice with disruption in a negative regulator of GH signaling (34
); (d) mice with liver-specific disruption of GH signaling (26
); and (e) mice with liver-specific deletion of Igf1
). Of these, all except mice in class b would be expected to have increased GH signaling in non-liver tissues, with an increase in lipolysis and at least some increase in levels of plasma FFA. However, of these, only the mice in class d have been shown to develop FL. This suggests that in addition to the increase in lipolysis and in levels of plasma FFA, a second hit is necessary to cause FL. Furthermore, this supports a model in which FL only develops when there is both an increase in the levels of FFA and a disruption in GH signaling in the liver. That is, while mice in class b have a decrease in GH signaling in the liver, they do not have an increase in lipolysis and therefore have normal or reduced levels of FFA. And indeed, as described above, these mice have increased adiposity, but no FL. On the contrary, mice in classes a, c, and e have increased levels of serum GH and intact GH signaling, with concomitant effects on lipolysis. They therefore have increased levels of plasma FFA, but have normal or increased GH signaling in the liver.
The evidence we have accumulated suggests that inhibition of GH signaling in the liver permits increased uptake of plasma FFA. This is apparent only when the levels of plasma FFA are increased. The data on de novo lipogenesis and TG secretion support this model, as while there is a general increase in the content of the FAs and glycerol in JAK2L livers, there is no increase in FA synthesis. In fact, there is a small decrease in the rate of palmitate synthesis. Furthermore, the ratio of oleate to stearate is extremely high, a fact that suggests an increase in stearoyl-CoA desaturase (SCD) activity (60
). Consistent with this, we observed significant increases in mouse SCD gene expression. Increases in SCD activity have indeed been reported in human and animals with FL (61
). Thus, while there is an increase in liver FA content, there is no increase in the rate of de novo synthesis, and this points strongly toward an increase in the uptake of plasma FA.
Fatty acid transport is thought to occur mainly through the family of slc27a fatty acid transport proteins as well as the scavenger receptor CD36 (62
). The 16-fold increase in expression of Cd36
in male JAK2L versus control liver was the sixth highest increase in the entire 25,000-gene Affymetrix array. Expression of CD36 is reported to be very low in normal mouse liver, and in our control samples, the expression at both the RNA and protein levels was indeed low. Prior reports have shown that increased expression of Cd36
can augment uptake of FA (64
). Therefore, we propose a model whereby the increased levels of CD36 in JAK2L livers augments uptake of plasma FFA as depicted in Figure . While the increase in Cd36
expression was partially attenuated in Lit-JAK2L mice, there was still a 10-fold increase in these animals (versus control), with normal levels of plasma FFA, suggesting that GH signaling itself represses expression of Cd36
. In addition, the level of Cd36
expression was very similar to that in Lit-Con mice.
Interestingly, the expression of Pparg
was increased dramatically in JAK2L liver and was increased to a similar degree in Lit-Con, Con-JAK2L, and Lit-JAK2L liver. CD36 is a well-known transcriptional target of PPARγ (65
). Previous work has implicated GH signaling (through STAT5b) in the transcriptional repression of PPARγ (46
). While GH was shown to repress the transcriptional activity of PPARγ, the effect did not appear to be mediated directly through a physical interaction between STAT5b and PPARγ (46
). Exactly how PPARγ is repressed by GH remains unclear, but one particularly intriguing idea is that this effect is mediated through HDAC3. Indeed, hepatocyte-specific deletion of HDAC3 resulted in de-repression of PPARγ and CD36 expression and in the accumulation of TG in the liver (54
). Treatment with a PPARγ inhibitor reduced expression of CD36 as well as the severity of steatosis in mice with hepatocyte-specific deletion of HDAC3 (54
). In JAK2L mice, treatment with GW9662 for 2 weeks reduced the expression of Cd36
in liver. These results support the hypothesis that transcriptional control of Cd36
is downstream of PPARγ. In addition, we have shown that by decreasing Cd36
expression in liver, the liver TG content is decreased. Overall, these data support our model in which the loss of GH signaling in JAK2L mice likely leads to a release of inhibition of Cd36
All of this suggests that abnormal GH signaling would predispose to development of FL in conditions where there are increased levels of plasma FFA, such as starvation (67
). Accordingly, administration of a small molecule JAK2 inhibitor would not be expected to cause FL unless there was concomitant increase in plasma FFA or if there was preferential drug uptake or action in the liver as one might expect with small molecule compounds. However, formal evaluation of the liver toxicity of these compounds remains to be determined. Furthermore, chronic inhibition of JAK2 might also be expected to result in increased visceral adiposity. The effect of JAK2 on GH-mediated lipolysis in adipocytes is of great interest for future study. Overall, these studies demonstrate what we believe to be a novel mechanism of FL and provide important insights into the possible effects of JAK2 inhibition in humans. At the core, the liver-specific deletion of JAK2 causes a remarkable redistribution of fat from peripheral stores to the liver. This may have implications for understanding the pathogenesis of NAFLD as well as the potential safety of JAK2 inhibition, but also suggests multiple lines of future research aimed at completely understanding the effect of GH signaling on fat metabolism and on lipid flux in hepatocytes.