Hepatocyte-specific, PPARγ-regulated mechanisms to promote steatosis in adult mice.

Peroxisome proliferator-activated receptor γ (PPARγ) is the target for thiazolidinones (TZDs), drugs that improve insulin sensitivity and fatty liver in humans and rodent models, related to a reduction in hepatic de novo lipogenesis (DNL). The systemic effects of TZDs are in contrast to reports suggesting hepatocyte-specific activation of PPARγ promotes DNL, triacylglycerol (TAG) uptake and fatty acid (FA) esterification. As these hepatocyte-specific effects of PPARγ could counterbalance the positive therapeutic actions of systemic delivery of TZDs, the current study used a mouse model of adult-onset, liver (hepatocyte)-specific PPARγ knockdown (aLivPPARγkd). This model has advantages over existing congenital knockout models, by avoiding compensatory changes related to embryonic knockdown, thus better modeling the impact of altering PPARγ on adult physiology, where metabolic diseases most frequently develop. The impact of aLivPPARγkd on hepatic gene expression and endpoints in lipid metabolism was examined after 1 or 18 weeks (Chow-fed) or after 14 weeks of low- or high-fat (HF) diet. aLivPPARγkd reduced hepatic TAG content but did not impact endpoints in DNL or TAG uptake. However, aLivPPARγkd reduced the expression of the FA translocase (Cd36), in 18-week Chow- and HF-fed mice, associated with increased NEFA after HF feeding. Also, aLivPPARγkd dramatically reduced Mogat1 expression, that was reflected by an increase in hepatic monoacylglycerol (MAG) levels, indicative of reduced MOGAT activity. These results, coupled with previous reports, suggest that Cd36-mediated FA uptake and MAG pathway-mediated FA esterification are major targets of hepatocyte PPARγ, where loss of this control explains in part the protection against steatosis observed after aLivPPARγkd.

Hepatic PPARγ Is Not Essential for the Rapid Development of Steatosis After Loss of Hepatic GH Signaling, in Adult Male Mice.

Our group has previously reported de novo lipogenesis (DNL) and hepatic triglyceride content increases in chow-fed male mice within 7 days of hepatocyte-specific GH receptor knockdown (aLivGHRkd). Here, we report that these changes are associated with an increase in hepatic expression of peroxisome proliferator-activated receptor γ (PPARγ), consistent with previous reports showing steatosis is associated with an increase in PPARγ expression in mice with congenital loss of hepatic GH signaling. PPARγ is thought to be an important driver of steatosis by enhancing DNL, as well as increasing the uptake and esterification of extrahepatic fatty acids (FAs). In order to determine whether hepatic PPARγ is critical for the rapid development of steatosis in the aLivGHRkd mouse model, we have generated aLivGHRkd mice, with or without PPARγ (ie, adult-onset, hepatocyte-specific double knockout of GHR and PPARγ). Hepatic PPARγ was not required for the rapid increase in liver triglyceride content or FA indexes of DNL (16:0/18:2 and 16:1/16:0). However, loss of hepatic PPARγ blunted the rise in fatty acid translocase/CD36 and monoacylglycerol acyltransferase 1 expression induced by aLivGHRkd, and this was associated with a reduction in the hepatic content of 18:2. These results suggest that the major role of PPARγ is to enhance pathways critical in uptake and reesterification of extrahepatic FA. Because FAs have been reported to directly increase PPARγ expression, we speculate that in the aLivGHRkd mouse, the FA produced by DNL enhances the expression of PPARγ, which in turn increases extrahepatic FA uptake, thereby further enhancing PPARγ activity and exacerbating steatosis overtime.

Islet insulin content and release are increased in male mice with elevated endogenous GH and IGF-I, without evidence of systemic insulin resistance or alterations in ß-cell mass.

UNLABELLED: It is clear that elevations in circulating GH can lead to an increase in insulin levels. This increase in insulin may be due to GH-mediated insulin resistance and enhanced lipolysis. However, there is also in vitro and in vivo evidence that GH acts directly to increase ß-cell proliferation and insulin production. Our laboratory recently developed an animal model with elevated endogenous GH levels associated with a small (25%), but significant, increase in IGF-I (HiGH mice). As expected, insulin levels were elevated in HiGH mice; however, whole body insulin sensitivity was not altered and glucose tolerance was improved. This metabolic phenotype suggests that modest elevations in circulating GH and IGF-I may enhance ß-cell mass and/or function, in the absence of systemic insulin resistance, thus improving glucose homeostasis. OBJECTIVE: To determine if ß-cell mass and/or function is altered in HiGH mice. DESIGN: Male HiGH mice and their littermate controls were fed a low-fat or high-fat diet. Body composition and circulating metabolic endpoints were monitored overtime. The pancreas was recovered and processed for assessment of ß-cell mass or in vitro basal and glucose-stimulated insulin secretion. RESULTS: HiGH mice showed elevated circulating insulin and normal glucose levels, while non-esterified FFA levels and triglycerides were reduced or normal, depending on diet and age. ß-cell mass did not differ between HiGH and control mice, within diet. However, islets from HiGH mice contained and released more insulin under basal conditions, as compared to control islets, while the relative glucose-stimulated insulin release did not differ. CONCLUSIONS: Taken together, these results suggest moderate elevations in circulating GH and IGF-I can directly increase basal insulin secretion without impacting ß-cell mass, independent of changes in whole body insulin sensitivity and hyperlipidemia.

Growth Hormone Inhibits Hepatic De Novo Lipogenesis in Adult Mice.

Patients with nonalcoholic fatty liver disease (NAFLD) are reported to have low growth hormone (GH) production and/or hepatic GH resistance. GH replacement can resolve the fatty liver condition in diet-induced obese rodents and in GH-deficient patients. However, it remains to be determined whether this inhibitory action of GH is due to direct regulation of hepatic lipid metabolism. Therefore, an adult-onset, hepatocyte-specific, GH receptor (GHR) knockdown (aLivGHRkd) mouse was developed to model hepatic GH resistance in humans that may occur after sexual maturation. Just 7 days after aLivGHRkd, hepatic de novo lipogenesis (DNL) was increased in male and female chow-fed mice, compared with GHR-intact littermate controls. However, hepatosteatosis developed only in male and ovariectomized female aLivGHRkd mice. The increase in DNL observed in aLivGHRkd mice was not associated with hyperactivation of the pathway by which insulin is classically considered to regulate DNL. However, glucokinase mRNA and protein levels as well as fructose-2,6-bisphosphate levels were increased in aLivGHRkd mice, suggesting that enhanced glycolysis drives DNL in the GH-resistant liver. These results demonstrate that hepatic GH actions normally serve to inhibit DNL, where loss of this inhibitory signal may explain, in part, the inappropriate increase in hepatic DNL observed in NAFLD patients.

MAP kinase phosphorylation is dispensable for cell division, but required for cell growth in Drosophila.

Proper activation of the Ras/MAPK pathway is broadly required during development, and in many cases, signal transduction downstream of the receptor is linear. Thus, different mechanisms exist to properly regulate the large number of specific developmental outputs that are required by the activation of this pathway. Previously, we have reported a regulated cytoplasmic sequestration of phosphorylated MAPK (pMAPK) in developing Drosophila compound eyes and wings "called MAPK Cytoplasmic Hold". In the developing wing, we have shown that cytoplasmic hold promotes the differentiation of wing vein tissue, while pMAPK nuclear translocation regulates growth and division. We had also suggested that the Ras pathway signals for inducing cell growth and cell division split upstream of the nuclear translocation of MAPK itself. Here, we further refine the role of MAPK in Drosophila. We report evidence that suggests, for the first time, that the phosphorylation of MAPK is itself another step in the regulation of cell growth and division in both Drosophila wing and eye cells. We show that inhibition of MAPK phosphorylation, or pMAPK nuclear translocation, is sufficient to block cell growth, but not cell division. These data suggest that non-phosphorylated MAPK is sufficient to induce cell division, but not cell growth, once inside the nucleus of the cell.

Delta and Egfr expression are regulated by Importin-7/Moleskin in Drosophila wing development.

Drosophila DIM-7 (encoded by the moleskin gene, msk) is the orthologue of vertebrate Importin-7. Both Importin-7 and Msk/DIM-7 function as nuclear import cofactors, and have been implicated in the control of multiple signal transduction pathways, including the direct nuclear import of the activated (phosphorylated) form of MAP kinase. We performed two genetic deficiency screens to identify deficiencies that similarly modified Msk overexpression phenotypes in both eyes and wings. We identified 11 total deficiencies, one of which removes the Delta locus. In this report, we show that Delta loss-of-function alleles dominantly suppress Msk gain-of-function phenotypes in the developing wing. We find that Msk overexpression increases both Delta protein expression and Delta transcription, though Msk expression alone is not sufficient to activate Delta protein function. We also find that Msk overexpression increases Egfr protein levels, and that msk gene function is required for proper Egfr expression in both developing wings and eyes. These results indicate a novel function for Msk in Egfr expression. We discuss the implications of these data with respect to the integration of Egfr and Delta/Notch signaling, specifically through the control of MAP kinase subcellular localization.