Glucocorticoid Receptor β Induces Hepatic Steatosis by Augmenting Inflammation and Inhibition of the Peroxisome Proliferator-activated Receptor (PPAR) α*♦

Glucocorticoids (GCs) regulate energy supply in response to stress by increasing hepatic gluconeogenesis during fasting. Long-term GC treatment induces hepatic steatosis and weight gain. GC signaling is coordinated via the GC receptor (GR) GRα, as the GRβ isoform lacks a ligand-binding domain. The roles of the GR isoforms in the regulation of lipid accumulation is unknown. The purpose of this study was to determine whether GRβ inhibits the actions of GCs in the liver, or enhances hepatic lipid accumulation. We show that GRβ expression is increased in adipose and liver tissues in obese high-fat fed mice. Adenovirus-mediated delivery of hepatic GRβ overexpression (GRβ-Ad) resulted in suppression of gluconeogenic genes and hyperglycemia in mice on a regular diet. Furthermore, GRβ-Ad mice had increased hepatic lipid accumulation and serum triglyceride levels possibly due to the activation of NF-κB signaling and increased tumor necrosis factor α (TNFα) and inducible nitric-oxide synthase expression, indicative of enhanced M1 macrophages and the development of steatosis. Consequently, GRβ-Ad mice had increased glycogen synthase kinase 3β (GSK3β) activity and reduced hepatic PPARα and fibroblast growth factor 21 (FGF21) expression and lower serum FGF21 levels, which are two proteins known to increase during fasting to enhance the burning of fat by activating the β-oxidation pathway. In conclusion, GRβ antagonizes the GC-induced signaling during fasting via GRα and the PPARα-FGF21 axis that reduces fat burning. Furthermore, hepatic GRβ increases inflammation, which leads to hepatic lipid accumulation.


Glucocorticoids (GCs) regulate energy supply in response to stress by increasing hepatic gluconeogenesis during fasting. Long-term GC treatment induces hepatic steatosis and weight gain. GC signaling is coordinated via the GC receptor (GR) GR␣, as the GR␤ isoform lacks a ligand-binding domain. The roles of the GR isoforms in the regulation of lipid accumulation is
unknown. The purpose of this study was to determine whether GR␤ inhibits the actions of GCs in the liver, or enhances hepatic lipid accumulation. We show that GR␤ expression is increased in adipose and liver tissues in obese high-fat fed mice. Adenovirus-mediated delivery of hepatic GR␤ overexpression (GR␤-Ad) resulted in suppression of gluconeogenic genes and hyperglycemia in mice on a regular diet. Furthermore, GR␤-Ad mice had increased hepatic lipid accumulation and serum triglyceride levels possibly due to the activation of NF-B signaling and increased tumor necrosis factor ␣ (TNF␣) and inducible nitric-oxide synthase expression, indicative of enhanced M1 macrophages and the development of steatosis. Consequently, GR␤-Ad mice had increased glycogen synthase kinase 3␤ (GSK3␤) activity and reduced hepatic PPAR␣ and fibroblast growth factor 21 (FGF21) expression and lower serum FGF21 levels, which are two proteins known to increase during fasting to enhance the burning of fat by activating the ␤-oxidation pathway. In conclusion, GR␤ antagonizes the GC-induced signaling during fasting via GR␣ and the PPAR␣-FGF21 axis that reduces fat burning. Furthermore, hepatic GR␤ increases inflammation, which leads to hepatic lipid accumulation.
A diet high in fat can lead to obesity and lipid accumulation in the liver, which increases proinflammatory cytokine production and the development of non-alcoholic fatty liver disease (NAFLD). 2 Also, NAFLD leads to peripheral tissue insulin resistance, possibly through a reduction of insulin clearance in the liver due to the build-up of lipids. NAFLD is characterized by hepatic fat accumulation that, when coupled with another "hit," such as increased oxidative stress, insulin resistance, or inflammation can lead to the development of non-alcoholic steatohepatitis (NASH) (1). The progression of hepatic steatosis may only represent the initial phase of several distinct deleterious pathways, and a "two-hit" theory has been used to explain the progression from NAFLD to NASH. The "first" hit is considered to be the accumulation of lipids in the liver. The "first hit" increases the vulnerability of the liver to other factors that may contribute to the "second hit" and promote hepatic injury and inflammation (2).
Interestingly, long-term glucocorticoid (GC) treatment, which is a known anti-inflammatory, induces hepatic steatosis and insulin resistance (3). The GC receptor (GR), is a complex single copy gene that is alternatively spliced to give rise to different isoforms ␣, ␤, ␥, A, and P (4). Due to direct binding, the GR␣ isoform is the protein responsible for the actions of GCs. GR␤, however, has a truncated helix 12 and is missing the ligand binding pocket known to bind to GCs (3). The known function of GR␤ is to act as a dominant-negative antagonist to GR␣ (3,5,(7)(8)(9). We have previously shown that GR␤ mRNA increased from fasting and refeeding in the livers of mice (8). Similarly, Dubois et al. (10) demonstrated that the rat GR␤ has a physiological role in the liver and rat GR␤ mRNA expression is increased in animals that have chronically elevated plasma insulin concentrations. Recently, He et al. (11) showed in a mouse with hepatic-specific GR knock-out and delivery of human GR␤-regulated gluconeogenesis and inflammation in liver. These studies suggest that GR␤ may have a role in the regulation of hepatic lipid storage and peripheral insulin resistance. However, the functions of the GR isoforms in the development of hepatic steatosis or insulin resistance remains to clarified.
During fasting, lipids that are taken up by the liver are a product of GC-induced lipolysis from adipose tissue (3). Typically, the glycerol released in adipose during fasting is used for gluconeogenesis in the liver for the production of new sugars from non-hexose substrates, which allows for the maintenance of blood glucose. The overstimulation of lipolysis or saturation of the blood with lipids from the diet leads to the accumulation of hepatic fat, de novo lipid synthesis, and inflammation. Lipid storage and the burning of fat are mediated by fat sensing nuclear receptors, PPAR␥ and PPAR␣, where PPAR␥ stores fat and PPAR␣ burns lipids by ␤-oxidation. Hepatic PPAR␣ increases the fat burning and glucose-lowering hormone, fibroblast growth factor 21 (FGF21), which causes a dramatic decrease in lipid accumulation in whole animals and sensitization to peripheral insulin signaling (12)(13)(14)(15)(16)(17)(18)(19). A reduction in PPAR␣ in the liver causes the development of fatty liver and inflammation (12,19). The GR␤ isoform has been shown to increase immune cell proliferation by inhibiting the anti-inflammatory actions of GCs (3,20), as well as increase inflammatory pathways in liver (11). Potentially, GR␤ may be involved in the initial phases of the development of NAFLD.
In this investigation, we found that elevated hepatic GR␤ increased lipid accumulation on a regular fat diet leading to elevated plasma glucose. Furthermore, we show that GR␤ decreased hepatic PPAR␣ expression resulting in a reduction of the hepatic FGF21 mRNA and plasma levels. Our data show that GR␤ may serve as the first hit in the progression of NAFLD.

GR␤ Expression Increases in
Response to HFD-Acute elevations in GC promote adipose tissue lipolysis and hepatic gluconeogenesis, which is orchestrated by GR␣. It has been proposed that obesity is a state of GC resistance that results in lipogenesis in adipose and the liver, leading to fatty liver disease (3). A probable mechanism for GC resistance in response to HFD is increased expression of GR␤. We have shown that GR␤ mRNA is increased during adipogenesis, whereas GR␣ expression was unchanged (21). In Fig. 1, A and B, we show that a HFD indeed increased GR␤ mRNA in adipose and liver, and this was accompanied by an elevation in transcript levels of the pro-inflammatory cytokine TNF␣. Neither GR isoform nor TNF␣ mRNA was altered in skeletal muscle in response to HFD (Fig. 1C), indicating that adipose and liver dysfunction may precede changes in skeletal muscle.
Overexpression of GR␤ in Mice Livers-Our data thus far indicate that under conditions of a HFD, GR␤ transcript levels increased in liver and adipose tissues, which correlated with elevated TNF␣ expression, an inflammatory cytokine that is associated with hepatic inflammation and fatty liver disease (22,23). To determine the role of GR␤ in hepatic lipid accumulation we constructed an adenovirus with mouse GR␤ cDNA (GR␤-Ad) and vector (vec-Ad). We next used the GR␤-Ad to overexpress GR␤ in the liver to determine whether elevated GR␤ expression is sufficient to induce hepatic steatosis. We infected the mice with GR␤-Ad or vec-Ad for 5 days on a regular fat diet. Overexpression of GR␤ was successfully targeted to the liver, without affecting adipose or muscle (Fig. 2, A-C). Importantly, GR␣ protein and mRNA levels were not affected in liver, muscle, or adipose (Fig. 2, A-C).
Overexpression of Hepatic GR␤ Increases Lipid Accumulation-Liver function is paramount in maintaining fat and glycogen storage, which also modulates normal circulating glucose levels. Recently Robert el al. (24) showed that in obese ob/ob mice the GR-Gilz pathway was suppressed in Kupffer liver cells, which caused lipid accumulation, suggesting that GR␣-directed paths are preventive in hepatic steatosis. He et al. (11) showed that adenoviral-associated virus (AAV) delivery of human GR␤ in mouse livers caused an increase in inflammatory pathways. However, they did not measure hepatic lipid content. Therefore, we wanted to determine whether increasing GR␤ in the livers of normal mice would cause an increase in hepatic lipid accumulation and possibly inflammatory markers. The histological assessment revealed increased Nile red staining for lipid accumulation in the liver of mice overexpressing GR␤ (Fig. 3A). Moreover, hepatic triglycerides (p Ͻ 0.05) (Fig.  3B) and serum triglycerides (p Ͻ 0.0001) (Fig. 3C) were markedly increased in liver overexpressing GR␤. Also, fatty acid synthase protein, but not mRNA, expression was significantly (p Ͻ 0.05) elevated in the liver of GR␤-Ad mice compared with vec-Ad, indicating increased de novo fat synthesis (Fig. 3D). Insulin resistance in the liver causes an increase in hepatic glucose production that results in hyperglycemia. Hepatic lipid accumulation is one mechanism underlying this response (25,26). Here we show that mice overexpressing GR␤ in the liver have elevated fasting glucose levels compared with vector-treated animals (Fig. 3E).
Hepatic Overexpression of GR␤ Suppresses Glycogen Storage-The liver serves as a primary reservoir for glycogen, which is used to maintain blood glucose levels during fasting. In livers overexpressing GR␤, glycogen content was reduced by 21%, but not significant, compared with Ad-vector-treated mice, which may be due to the reduction in expression of glycogen synthase 2 (Gys2) (Fig. 4A). Gys2 stores glucose as glycogen in the liver for use during times of fasting and GSK3␤ phosphorylates Gys2 to reduce glycogen storage. Phosphorylation of serine 9 of GSK3␤ inhibits kinase activity, which typically occurs during feeding. The phosphorylation of serine 9 of GSK3␤ was significantly lower (p Ͻ 0.01) in GR␤-Ad mice indicating activation (Fig. 4B). Additionally, the expression of the gluconeogenic genes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase was reduced in liver overexpressing GR␤ (Fig. 4C). However, Foxo1, which also regulates gluconeogenesis, was unchanged. Taken together, this indicates that GR␤ causes hepatic lipid accumulation and possibly interferes with glycogen storage in long-term high GR␤ levels.
We previously demonstrated that GR␤ regulates cell growth through an Akt1-PTEN dependent signaling (9). Here we show that hepatic PTEN is suppressed when GR␤ is overexpressed in the liver (Fig. 4D), consistent with our previous findings (26).
However, Akt1 expression was not altered. On the other hand, GR␤ reduced hepatic Akt2 expression. Akt2 has been shown to regulate metabolic function, particularly glucose homeostasis (27). Akt increases glycogen storage by inhibition of GSK3␤ through phosphorylation of serine 9. GR␤ may serve to increase lipid accumulation during times of fasting and reduce glycogen storage to inhibit gluconeogenesis and hepatic glucose production.
Hepatic Overexpression of GR␤ Increases NF-B Activity-GR␤ has recently been shown to cause inflammation in the liver (11). However, the role of GR␤ in the regulation of M1 proinflammatory or M2 anti-inflammatory macrophages or the direct effect of GR␤ on NF-B phosphorylation or transcriptional activity is unknown. The NF-B signaling pathway con-tributes to the regulation of inflammation, particularly by increasing the production of TNF␣. Also, NF-B expression is increased in livers with inflammation and steatosis (22,28). In Fig. 5A we show that NF-B serine 536 phosphorylation is elevated and the protein and mRNA expression of IB␣, an inhibitor of NF-B, was suppressed in livers overexpressing GR␤. No significant change was observed in F480 total macrophage marker expression (Fig. 5B). However, GR␤ overexpressing livers had higher expression of proinflammatory M1 macrophage markers TNF␣ and inducible nitric-oxide synthases (iNOS), and reduced anti-inflammatory M2 macrophage markers arginase 1 (Arg-1) and FIZZ1 (Fig. 5C). Even though there was no change in total macrophage marker, these results suggest that GR␤ enhanced proinflammatory macrophages and reduced anti-inflammatory M2. Furthermore, we demonstrated that GR␤, but not GR␣ increased the promoter activity of NF-B (Fig. 5, D and E), indicating that overexpression of GR␤ in the liver enhances NF-B activity in the pathogenesis of hepatic steatosis. Last, the promoter activity of the tumor suppressor PTEN was decreased in response to GR␤, which is consistent with our previous findings (7,9). NF-B has also previously been shown to reduce PTEN expression (29).
Here we show that NF-B and GR␤ together reduced the PTEN promoter more significantly (p Ͻ 0.05) than either transcription factor alone. In Fig. 5C, we show that GR␤ significantly increased NF-B activity, and this effect was lost with GR␤ alone. GR␣ had no effect on NF-B activity at the NF-B-luc promoter but did significantly (p Ͻ 0.05) decrease the promoter without overexpression of p65. Inter-estingly, GR␣ attenuated NF-B suppression of the PTEN promoter and increased the PTEN-luc. The overexpression of GR␤ in mice livers caused a shift in macrophages to the pro-inflammatory M1 population, which combined with enhanced NF-B activation leads to hepatic lipid accumulation and steatosis.
Hepatic GR␤ Suppresses PPAR␣-mediated Signaling-PPAR␣ is a nuclear receptor that has been shown to attenuate hepatic lipid accumulation, and is reduced in obesity (9, 12, 30 -34). Also, PPAR␣ is increased in the liver during fasting and reduced during feeding (35). Interestingly, GR␤ overexpression in mice livers decreased PPAR␣ protein and mRNA expression on a normal diet (Fig. 6, A and B). PPAR␣ has been shown to increase genes in the ␤-oxidation pathway to reduce lipid accumulation. Also, PPAR␣ increases the hormone fibroblast growth factor 21 (FGF21) in the liver to be excreted to affect glucose and lipid storage in adipose and liver (12, 14, 17, 19, 36 -39). FGF21 is increased by PPAR␣ during fasting, which elevates AMP-activated protein kinase (AMPK) and glucose transporter 1 (Glut1) for energy metabolism and glucose usage (15, 17-19, 36, 38, 40). The FGF21 protein and mRNA were decreased in GR␤-Ad livers and serum (Fig. 6, A and B). Additionally, the FGF21-regulated gene, Glut1, was reduced in livers with overexpressed GR␤. Interestingly, Glut1 and AMPK mRNA expression, but not FGF21, was also decreased in adipose tissue of the GR␤-Ad mice, although GR␤ expression was not increased. The reduction in serum FGF21, which originates from the liver (40), also caused decreased signaling in adipocytes.
We then overexpressed GR␤ or GR␣ versus PPAR␣ and measured the activity at the minimal PPAR promoter PPRE-3tk-Luc to determine whether they regulate PPAR␣ transcriptional activity. GR␤ significantly decreased PPAR␣ transcriptional activity with vehicle (p Ͻ 0.01) and PPAR␣ agonist WY-14,643 by over 50% (p Ͻ 0.0001) (Fig. 7A). Conversely, GR␣ overexpression caused a significant (p Ͻ 0.01) increase in PPAR␣ transcriptional activity. Glucocorticoids have been shown to regulate FGF21 expression in a feed-forward loop (41). To determine whether GR␤ or GR␣ can regulate FGF21 expression, we measured luciferase activity of the FGF21 promoter construct (FGF21-Luc) with GR␤ or GR␣ overexpressed. Not surprisingly, GR␣ significantly increased FGF21 promoter activity (Fig. 7B). However, GR␤ alone had no effect. To determine whether GR␤ or GR␣ regulate PPAR␣ transcriptional activity at the FGF21 promoter, we treated cells overexpressing GR␤ or GR␣ with WY-14,643. Interestingly, GR␤ inhibited PPAR␣ activity by 26% at the FGF21 promoter (p Ͻ 0.001), but GR␣ had no effect (Fig. 7C).

Discussion
We show in this study that GR␤ increases lipid storage in the liver, and functions as the precursor for hepatic inflammation, which may act as the first hit in the progression of NAFLD. Inflammation in the liver serves as a precursor for several deleterious events that lead to hepatic lipid accumulation and NAFLD (19,34,(42)(43)(44)(45). The liver does secret TNF␣, which causes infiltration of the immune system and is considered one of the many hits in the first stages of NAFLD (46). To further determine the specific role of GR␤ in the development of hepatic steatosis, we developed a model of increased hepatic expression using an adenoviral approach. Recently, He et al. (11) showed that an AAV-mediated delivery of human GR␤ in mice caused increased inflammation. Furthermore, Warrier et al. (45) demonstrated that mice with glucocorticoid resistance are susceptible to diet-induced hepatic steatosis and inflammation. In our murine model, hepatic-specific viral overexpres-sion of mouse GR␤ resulted in the augmentation of liver fatty acid production as evidenced by increased serum and hepatic triglycerides, and alterations in proteins associated with fatty acid synthesis and marked hepatic steatosis, which occurred in a very short (5 day) time frame and on a regular fat diet. In addition, hepatic overexpression of GR␤ resulted in hyperglycemia without significant changes in insulin levels, which sug- gests alterations in gluconeogenesis or reduced hepatic insulin signaling due to increased lipids. All of these factors are altered in the obese and those with metabolic disorders.
Our results also demonstrate that diet-induced obese mice have a selective increase in GR␤ expression in adipose and the liver without any changes in the levels of GR␣ in these tissues. The increase in GR␤ expression was also associated with an elevation in the levels of the pro-inflammatory cytokine TNF␣. In addition to our group, others have shown that TNF␣ and proinflammatory cytokines increase GR␤ expression (8,(47)(48)(49). TNF␣ levels are known to be higher in the obese (50,51). However, if TNF␣ induces GR␤ in obesity is unknown. We did show in this investigation that overexpression of GR␤ in mouse livers caused an increase in TNF␣ and iNOS, which are M1 proinflammatory macrophage markers, with no change in total macrophages (F480). GR␤ may drive the infiltration of the immune system in the liver, which is a known characteristic of the onset of NAFLD. Because high-fat feeding induces inflammation and possibly GC resistance (3), it is likely that increased expression of GR␤ contributes to these phenotypes in response to an increase in high-fat intake. Although a high-fat diet increased GR␤ levels in both adipose and liver, the levels of GR␤ were not altered in muscle suggesting that skeletal muscle may not contribute to the first stages of high-fat diet-induced hepatic steatosis. Interestingly, the findings of He et al. (11) for the AAV-mediated delivery of human GR␤ showed a reduced plasma glucose level, which is a conundrum because inflammation in the liver leads to glucose intolerance and high circulating insulin levels (42)(43)(44)(45)(52)(53)(54). Furthermore, in the study by He et al. (11) hepatic lipid content was not measured, even though inflammation was present and is the precursor to fatty acid production in the liver (19,34,(42)(43)(44)(45). Our observation that overexpression of mGR␤ caused an increase in M1 proinflammatory macrophage markers TNF␣ and iNOS as well as phosphorylation of NF-B agree with these findings. However, we found significantly higher glucose levels and hepatic lipid content, which was not observed with the human GR␤ overexpression. GR␤ activation of NF-B may be an important first step in the progression of NAFLD to NASH. However, more investigations on the roles of GR␤ and GR␣ are needed to understand their involvement in hepatic lipid accumulation.
Hepatic steatosis is the initial step in the progression of NAFLD to the development NASH, which is believed to occur through multiple parallel hits on the liver with the first hit being the accumulation of lipids. The second hit, which increases the vulnerability of the liver to the development of NASH can be another factor such as increased oxidative stress or inflammation (55). To this end, hepatic overexpression of GR␤ not only resulted in steatosis but also resulted in increased inflammation as demonstrated by increases in both NF-B and proinflammatory cytokine TNF␣. GR␤ increased NF-B phosphorylation levels possibly by down-regulating IB␣, inhibition of GR␣, or possibly through direct binding of GR␤-NF-B heterodimers on promoters. NF-B is a master transcription regulator of several genes involved in inflammation, and GR␤ inhibition of GCs may play an important role. GR␤ enhanced NF-B activity at the NF-B minimal and PTEN promoters. Progression from NAFLD to NASH can be accelerated via the loss of the tumor suppressor PTEN (56). GR␤ overexpression resulted in a significant decrease in hepatic PTEN levels suggesting that downregulation of this pathway may be another contributing factor to the development of hepatic steatosis in this model.
GR␤ may serve to decrease the GR␣-PPAR␣ axis, which has been shown to be necessary for hepatic lipid catabolism (57). PPAR␣ increases genes involved in the ␤-oxidation pathway, which cause the burning of fat. PPAR␣ levels are significantly lower in the obese (19) and are increased during fasting and reduced with feeding (35). GR␤ may be involved in the obesityinduced or feeding reduction of PPAR␣ expression, as overexpression significantly decreased hepatic levels and we also showed it reduced transcriptional activity at the minimal PPRE and FGF21 promoters. It is interesting to note that GR␤ overexpression results in both enhanced GSK3␤ activity (decreased GSK3␤ phosphorylation) as well as lower levels of PPAR␣ and its target gene, FGF21. PPAR␣ also binds to the promoter of GYS2 to increase expression (58), which together interacts to regulate glycogen storage and steatosis. GSK3␤ is a negative regulator of GYS2 and decreases activity, causing alterations in hepatic glycogen production. We found reduced glycogen, but not significant, in the mice of this study, which were fasted and would have been producing glucose by glycogenolysis. Future studies on fed mice may show a significant reduction in glycogen during feeding with high GR␤ levels, which is when glycogen is stored. GR␤ overexpression resulted in a significant decrease in the levels of phosphorylation of serine 9 in GSK3␤, which increases its' activity. The decrease in GSK3␤ phosphorylation could be due to the lower Akt2 levels observed in the liver of GR␤ overexpressing mice, as previous studies have demonstrated the regulation of GSK3␤ phosphorylation by the Akt pathway (59,60).
GR␤ overexpression resulted in a decrease in both the hepatic and serum levels of the PPAR␣ target gene, FGF21. FGF21 is a hepatic hormone that has autocrine and exocrine effects that impact hepatic glycogen storage and reduces steatosis (Fig. 8) (12,15,16,19,38). It can also improve glycemic control and the management of body weight (13, 15-17, 19, 37-39, 61, 62). FGF21 expression is enhanced by both GR␣ and the PPAR␣ agonist WY-14,643 (WY). However, GR␤ overexpression attenuated WY-14,643-induced FGF21 activation without any significant effect on basal FGF21 promoter activity. Mice lacking FGF21 have hepatic insulin resistance and increased glucose production from the liver (63), which may indicate that GR␤ regulation of PPAR␣ at the FGF21 promoter may be the first step in the cascade of events that lead to hepatic lipid accumulation. These results suggest that GR␤ regulates FGF21 in a PPAR␣-dependent fashion. In support of this hypothesis, GR␤ overexpression led to a decrease in both protein and mRNA levels of PPAR␣ in the liver. Furthermore, GR␤ overexpression attenuated both basal and WY-14,643-mediated increases in PPAR␣ activity in COS7 cells. Together, these data may indicate that GR␤ increases during feeding to reduce PPAR␣ expression. However, the response of GR␤ or GR␣ to fasting is unknown.
In conclusion, our results demonstrate that high-fat feeding specifically increases GR␤ expression in adipose and liver but not in muscle. Liver-specific overexpression of GR␤ led to hyperglycemia and hepatic steatosis. However, others have shown that the human GR␤ caused a reduction in blood glucose levels in mice, even though inflammation was present. A stable hepatic-specific overexpression of GR␤ in mice would help reveal its role in glucose mediation. Enhanced expression of GR␤ decreases hepatic PPAR␣ levels and its' target gene, FGF21, whereas enhancing inflammation through the downregulation of IB␣ and increased levels of NF-B and TNF␣. PPAR␣ and FGF21 are known regulators of hepatic fat and blood glucose levels (19,34). GR␤ overexpression also decreased hepatic levels of PTEN, which has been previously associated with the development of cancer and NASH. Longterm GR␤ overexpression in mice may lead to NAFLD, NASH, and possibly cirrhosis or cancer. These results highlight the potential for alterations in GR␤ activity to result in hepatic steatosis and its possible role in the progression of NAFLD to NASH. Targeted therapeutics directed specifically against GR␤, such as Sweet-P (5, 64), could provide new remedies for the treatment of NAFLD or NASH.

Animals
The experimental procedures and protocols of this study conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Insti-tutional Animal Care and Use Committee at the University of Toledo in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Animal activity and grooming were monitored daily to assess overall animal health. Animals were housed in a temperature-controlled environment with 12-h dark-light cycle.

Regular and High Fat Diets
The breeding colony was maintained on standard chow (regular diet) containing 12 kcal % fat ad libitum. Experimental mice were separated into two groups of 8-week-old males. One group was fed (ad libitum) a regular diet for 4 weeks, and the second was fed (ad libitum) a HF diet containing 45 kcal % fat (catalogue number D12451; Research Diets, New Brunswick, NJ). The HF regimen chosen is known to induce borderline to moderate steatosis and insulin resistance in most strains of mice, without stimulating hepatic inflammation (65).

Adenoviral Studies
All mice were 8-week-old male C57BL/6 that were purchased from Jackson Labs. Mice were individually caged in specific pathogen-free enclosures with a 12-h light/dark cycle at 22 to 24°C. All animals were fed a regular chow diet and injected with adenovirus as described below.

Adenovirus Construction and Liver Overexpression
To develop the adenovirus, mouse GR␤ cDNA was inserted into BglII and XbaI restriction sites in the pAdTrack-CMV vector and the pAd-Easy system was used to build the virus as defined in Ref. 66. The adenoviruses were multiplied in 293 HEK cells and purified using the AdEasy Virus Purification Kit (Agilent Technologies, catalogue number 240244-1). Either adenovirus overexpression GR␤ (GR␤-Ad) or empty vector (vec-Ad) were injected by tail vein in mice. All tail vein injections were performed in a dedicated BSL2-certified animal holding room. Mice were anesthetized with inhaled isoflurane (1.5 to 2.5%) while on a warming pad. Although anesthetized, a warm pack was wrapped around the tail to cause vessel dilation. The tail was gently pulled down and a syringe was inserted into the tail vein. Animals remained in BSL2 housing for the duration of the study following injection (5 days). All adenovirusinfected mice were feed a regular diet chow (as described above) during the 5-day study. Mice were fasted overnight and then tissues and blood were harvested for analysis.

Serum Analysis
At the termination of the study, mice were fasted overnight and then euthanized. Blood was extracted from all animals for measurement of glucose, insulin, and FGF21. For glucose measurements ϳ3 to 5 l were analyzed for blood glucose concentration using the laboratory animal-specific Alpha Trak glucometer and strips (Abbott Labs). The insulin concentrations were determined in serum samples of GR␤-Ad and vec-Ad mice using a commercially available ELISA as described by the provider (Crystal Chem) and as previously described (67). Serum triglycerides were determined with commercially available reagents (Pointe Scientific). The lower limit of detection for serum triglycerides was 5 mg/dl. FGF21 serum concentra-  DECEMBER 9, 2016 • VOLUME 291 • NUMBER 50

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tions from mice fasted overnight were determined using an ELISA (Millipore). The lowest level of FGF21 detected by this test is 49.4 pg/ml. All samples were run in duplicate and diluted to fit within the respective standard curve. Samples were re-assayed if the coefficient of variance was greater than 10% for duplicates.

Liver Glycogen
Liver glycogen content was determined from fasted mice using a commercially available kit (Abnova). Briefly, 10 mg of tissue was homogenized in 200 l of dH 2 O using the Tissue Lyser bead homogenizer (Qiagen). Samples were boiled for 5 min and centrifuged at 13,000 rpm for 5 min. Then, 50-l samples and standards were added to a 96-well plate and developed according to the manufacturer's instructions. Samples were run in duplicate and samples were re-assayed if the coefficient of variance was greater than 10% or samples were outside the detectable limits of the test (0.0004 to 2 mg/ml), and multiplied by the appropriated dilution factor for actual concentrations.

Transfection and Promoter Luciferase Reporter Assays
Transient Transfection-Cells were plated on a 6-well dish in DMEM containing 10% calf serum prior to transfection and allowed to grow to 85-90% confluence. Cells were washed with Opti-MEM and transfected using GeneFect (Alkali Scientific, Inc.), according the manufacturer's protocol. Opti-MEM was removed after 5 h and DMEM containing 10% FBS/serum was added.
Promoter Reporter Assays-Expression vector for mGR␤ (pMGR␤-H57) was constructed as previously described (8). NF-B minimal promoter-luciferase and PTEN promoter-luciferase activity was measured by firefly luciferase in the presence of the p65 subunit of NF-B versus GR␤ and/or GR␣ (PTEN-luc was made as defined in Ref. 6), and the pRL-CMV Renilla reporter for normalization to transfection efficiency. PPAR␣ minimal promoter PPRE-3tk-luciferase and FGF21promoter-luciferase (gift from Dr. David Mangelsdorf at the University of Texas Southwestern) activity were measured by firefly luciferase in the presence of PPAR␣ versus GR␤ and/or GR␣, and the pRL-CMV Renilla reporter for normalization to transfection efficiency. Transient transfection was achieved using GeneFect (Alkali Scientific, Inc.). Twenty-four-h posttransfected cells were treated for 24 h in dialyzed fatty acid-free FBS and then lysed. The luciferase assay was performed using the Promega dual luciferase assay system (Promega, Madison, WI).

Quantitative Real-time PCR Analysis
Total RNA was harvested from mice by lysing tissues using a Qiagen Tissue Lyser LT (Qiagen) and then extraction by a 5-Prime PerfectPure RNA Tissue Kit (Fisher Scientific Company, LLC). Total RNA was read on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR amplification of the cDNA was performed by quantitative real-time PCR using TrueAmp SYBR Green qPCR SuperMix (Alkali Scientific). The thermocycling protocol consisted of 5 min at 95°C, 40 cycles of 15 s at 95°C, and 30 s at 60°C and finished with a melting curve ranging from 60 to 95°C to allow distinction of specific products. Normalization was performed in separate reactions with primers to GAPDH mRNA.

Statistical Analysis
Data were analyzed with Prism 6 (GraphPad Software, San Diego, CA) using analysis of variance combined with Tukey's post-test to compare pairs of group means or unpaired t tests. Results are expressed as mean Ϯ S.E. Additionally, one-way analysis of variance with a least significant difference post hoc test was used to compare mean values between multiple groups, and a two-tailed, and a two-way analysis of variance was utilized in multiple comparisons. p values of 0.05 or smaller were considered statistically significant.