Antisense Protein Tyrosine Phosphatase 1B Reverses Activation of p38 Mitogen-Activated Protein Kinase in Liver of ob/ob Mice

Rebecca J. Gum, Lori L. Gaede, Matthew A. Heindel, Jeffrey F. Waring, James M. Trevillyan, Bradley A. Zinker, Margery E. Stark, Denise Wilcox, Michael R. Jirousek1, Cristina M. Rondinone and Roger G. Ulrich2

Abbott Laboratories, Abbott Park, Illinois 60064

Address all correspondence and requests for reprints to: Rebecca J. Gum, Abbott Laboratories, Department R-4CK, AP10-1, 100 Abbott Park Road, Abbott Park, Illinois 60064-3502. E-mail: rebecca.gum{at}abbott.com.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Phosphorylation of stress-activated kinase p38, a MAPK family member, was increased in liver of ob/ob diabetic mice relative to lean littermates. Treatment of ob/ob mice with protein tyrosine phosphatase 1B (PTP1B) antisense oligonucleotides (ASO) reduced phosphorylation of p38 in liver—to below lean littermate levels—and normalized plasma glucose while reducing plasma insulin. Phosphorylation of ERK, but not JNK, was also decreased in ASO-treated mice. PTP1B ASO decreased TNF{alpha} protein levels and phosphorylation of the transcription factor cAMP response element binding protein (CREB) in liver, both of which can occur through decreased phosphorylation of p38 and both of which have been implicated in insulin resistance or hyperglycemia. Decreased p38 phosphorylation was not directly due to decreased phosphorylation of the kinases that normally phosphorylate p38—MKK3 and MKK6. Additionally, p38 phosphorylation was not enhanced in liver upon insulin stimulation of ASO-treated ob/ob mice (despite increased activation of other signaling molecules) corroborating that p38 is not directly affected via the insulin receptor. Instead, decreased phosphorylation of p38 may be due to increased expression of MAPK phosphatases, particularly the p38/ERK phosphatase PAC1 (phosphatase of activated cells). This study demonstrates that reduction of PTP1B protein using ASO reduces activation of p38 and its substrates TNF{alpha} and CREB in liver of diabetic mice, which correlates with decreased hyperglycemia and hyperinsulinemia.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TYPE 2 DIABETES is characterized by insulin resistance in tissues such as liver, fat, and skeletal muscle (1). A variety of research efforts into type 2 diabetes are directed at increasing insulin sensitivity by stimulating insulin-dependent signaling pathways. An example of one such research target is protein tyrosine phosphatase 1B (PTP1B; Ref. 1). PTP1B is thought to negatively regulate insulin signaling by dephosphorylating the insulin receptor (IR) as well as its substrates IRS-1 and IRS-2 (2, 3, 4). PTP1B protein levels are increased in insulin-resistant, diabetic patients (5). In addition, nondiabetic mice lacking the PTP1B gene (PTP1B-/- mice) exhibit increased insulin sensitivity, resistance to weight gain on a high fat diet, and increased basal metabolic rate with no apparent deleterious effects (6, 7). Thus, inhibition of PTP1B is expected to increase insulin-dependent signaling and insulin sensitivity in diabetic models. Indeed, we have recently shown increased insulin-dependent signaling in liver of ob/ob mice treated with PTP1B antisense oligonucleotides (ASO; Refs. 8, 9).

However, in addition to regulating glucose uptake and production, insulin also regulates hepatocellular growth and inflammation via the MAPKs (10). Thus, one concern with increased insulin sensitivity and signaling is the potential for increased mitogenic and inflammatory effects possibly leading to hepatic hypertrophy, hyperplasia, or inflammation (10, 11, 12). MAPKs are a family of serine-threonine protein kinases involved in both stress and mitogenic responses to stimuli (12, 13, 14). There are at least three families of MAPKs/ERKs (15), p38 MAPKs (14, 16, 17, 18), and Jun amino terminal kinases (JNKs; Ref. 19). Both ERK and p38 are responsive to growth factors such as insulin, whereas p38 is also activated by stress and inflammatory agents (12, 14, 15, 17, 20) and is often referred to as a stress-activated protein kinase. The IR can activate ERK through a signaling cascade involving Src homology containing protein (SHC) or IRS complexed with growth factor receptor bound 2 (GRB2) and son of sevenless (SOS), leading to sequential activation of Ras, Raf1, MAPK/ERK kinase 1/2 (MEK1/2), and ERK1/2 (21, 22). The pathway from the IR to p38 is more controversial but is expected to occur through activation of MAPK kinase 3 (MKK3) or MKK6 (MAPK kinases or MAPKKs), the kinases which are known to activate p38 in response to other stimuli (12, 14, 23, 24), possibly via a Rac-, Rho-, or Cdc42-dependent mechanism (23).

Activated MAPK can then phosphorylate a variety of downstream substrates. TNF{alpha} and cAMP response element binding protein (CREB) are two such substrates of p38 (12, 14, 25, 26). TNF{alpha} protein levels are increased by p38 via a posttranscriptional mechanism involving release of a secondary RNA structure of TNF{alpha}, which then allows translation to proceed (12, 27). TNF{alpha} can also activate p38 and JNK and can increase production of numerous cytokines, thus eliciting an immune response (14, 27). In addition, activation of TNF{alpha} has been associated with insulin resistance and inflammatory responses in type 2 diabetes (28, 29, 30). CREB regulation is controlled both at the level of transcription as well as posttranslationally via phosphorylation (31). Both ERK and p38 can phosphorylate CREB via several downstream kinases including MSK1 for ERK and p38, p90RSK for ERK, and MAPKAPK2 for p38 (12, 14, 26). CREB, in turn, controls or influences transcription of a number of genes including basal transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting step for gluconeogenesis in liver (31, 32, 33, 34).

The activation status of MAPK in liver, a tissue critically involved in diabetes, has not been examined previously. In this study, we sought to determine the status of MAPK phosphorylation in liver of diabetic ob/ob mice and the effects of reducing PTP1B protein, using PTP1B ASO, on MAPK activity. We were initially concerned that enhanced insulin signaling and increased insulin sensitivity, particularly if constitutive, would lead to adverse effects via increased activation of MAPK. Instead, we found increased, constitutive phosphorylation of p38, and to a lesser degree ERK, in ob/ob diabetic mice relative to their more normal, ob/+, lean littermates. Treatment of ob/ob mice with PTP1B ASO reduced the basal activation state of p38 and ERK to levels at or below lean littermate levels. Substrates of p38 were also affected. TNF{alpha} protein levels and phosphorylation of CREB were increased in ob/ob mice and decreased in PTP1B ASO-treated mice. The decrease in p38 phosphorylation with PTP1B ASO was not due to decreased phosphorylation of the MAPK kinases MKK3, MKK6, or MKK4 but rather may have been due, at least in part, to increased levels of MAPK phosphatases (MKPs) particularly PAC1 (phosphatase of activated cells), a phosphatase that specifically dephosphorylates p38 and ERK but not JNK (35). These data, along with previous reports of p38 activation in aorta and glomeruli of diabetic rats (36, 37), implicate p38 activation as a common and important factor in diabetes and its complications.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Phosphorylation of p38 and ERK MAPK Is Increased in Liver of ob/ob Diabetic Mice Relative to Lean Littermates, and This Is Reversed by PTP1B ASO Treatment
Western blotting for phosphorylated (active) p38, ERK and JNK was performed on liver from saline control-treated and PTP1B ASO-treated diabetic ob/ob mice to investigate whether treatment with an insulin-sensitizing agent such as PTP1B ASO would constitutively increase MAPK activation in liver either through increased insulin stimulation or increased stress responses. Instead p38, and to a lesser extent ERK1, were found to be constitutively activated in liver of diabetic ob/ob mice relative to lean ob/+ mice (Fig. 1Go, A and B, respectively). Rather than increasing p38 or ERK phosphorylation, PTP1B ASO treatment reduced the phosphorylation of both p38 and ERK in liver of ob/ob mice relative to saline control-treated mice (Fig. 1Go, A and B, respectively). The decrease in phosphorylation of both kinases upon PTP1B ASO treatment was to levels at or below those observed with lean, ob/+ littermates. Phosphorylation of another MAPK, JNK, was not statistically different in liver of ob/ob vs. lean mice and was not affected by PTP1B ASO treatment (Fig. 1CGo). In a separate experiment, treatment of ob/ob mice with 50 mg/kg PTP1B ASO twice weekly for 3 wk also resulted in decreased phosphorylation of hepatic p38, whereas similar treatment with a control oligonucleotide did not significantly affect hepatic p38 activation (Fig. 1DGo), indicating that decreased basal activity of p38 in PTP1B ASO-treated mice was not due to nonspecific oligonucleotide effects.



View larger version (45K):
[in this window]
[in a new window]
 
Figure 1. Phosphorylation of p38 and ERK But Not JNK Is Decreased in Liver of ob/ob Mice Treated with PTP1B ASO

Western blots and quantitation for phospho-p38 (A), phospho-ERK (B), and phospho-JNK (C) using liver extracts from mice treated with saline control or PTP1B ASO (25, 2.5, and 0.25 mg/kg). D, Results from a previous experiment in ob/ob mice comparing phosphorylation of p38 in PTP1B ASO-treated ob/ob mice relative to saline control- and oligo control-treated ob/ob mice using 50 mg/kg instead of 25 mg/kg of the oligos. The top blot in each panel (i.e. marked "P-p38" in panel A) is a phospho-specific antibody blot, whereas the lower blot (i.e. marked "p38" in panel A) is the whole protein blot of the same protein. Quantitation, below the blots, shows changes in the phosphorylated protein relative to the ob/ob saline control group for panels A–C and relative to the PTP1B ASO treated group for D. The data are expressed as average ± SEM (*, P < 0.05; **, P < 0.01; ***P < 0.001 using a two-tailed Student’s t test).

 
Decreased p38 Phosphorylation Correlates with Decreased PTP1B Protein in Liver, and with Decreased Plasma Glucose Levels in PTP1B ASO-Treated ob/ob Mice and in Lean ob/+ Mice
The decrease in p38 phosphorylation correlated with the decrease in PTP1B protein in liver of ob/ob mice treated for 6 wk with PTP1B ASO (Fig. 2AGo), and similar low levels of p38 phosphorylation and PTP1B protein were observed in lean ob/+ mice (Figs. 1AGo and 2AGo, respectively; PTP1B protein correlation relative to phospho-p38, r = 0.81 for all groups including lean mice and r = 0.97 for ob/ob saline control- and PTP1B ASO-25-treated mice). Both the decrease in p38 phosphorylation and the decrease in PTP1B protein correlated with the decrease in plasma glucose at the two higher doses of treatment (r = 0.76 and 0.62, respectively) and with the plasma glucose level in lean ob/+ mice (Fig. 2BGo). The decrease in p38 phosphorylation and PTP1B protein occurred before a decrease in plasma glucose at the lowest dose of PTP1B ASO, indicating that these events may precede glucose lowering. This implies that the decrease in p38 phosphorylation is not caused by normalization of plasma glucose. The decrease in glucose appeared to precede the decrease in insulin with the normalization of glucose occurring within the first 2 wk of the study at the 25 mg/kg dose of PTP1B ASO, whereas insulin levels continued to decrease for the first 4–5 wk of the study at the same dose (9). Likewise, the decrease in insulin correlates with the decrease in PTP1B protein and the decrease in p38 phosphorylation at the 25-mg/kg dose of PTP1B ASO and with the lean ob/+ animals but appears to lag behind at the lower doses of PTP1B ASO (Fig. 2CGo). In addition, the 25 mg/kg PTP1B ASO-treated ob/ob mice exhibited increased sensitivity to insulin as determined by insulin and glucose tolerance tests as well as increased activation of insulin-dependent signaling pathways (8, 9).



View larger version (19K):
[in this window]
[in a new window]
 
Figure 2. PTP1B Protein, Plasma Glucose, and Insulin Are Decreased in PTP1B ASO-Treated ob/ob Mice to Levels Comparable to Lean, ob/+ Littermates

PTP1B protein (A) as detected by Western blotting using liver extracts from ob/ob or lean (ob/+) mice treated with saline control or PTP1B ASO (25, 2.5, and 0.25 mg/kg, twice weekly for 6 wk; Refs. 9 and 45 ). Quantitation of PTP1B protein (A) is shown relative to the ob/ob saline control. Nonfasting plasma glucose (B) and insulin (C) levels are shown as detected at the end of 6 wk of PTP1B ASO or saline-control treatment of ob/ob or ob/+ mice. Data for all experiments are expressed as the average ± SEM. The level of significance is given as a two-tailed Student’s t test, where *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Substrates of p38: Decreased Phosphorylation of CREB and Decreased Protein Levels of TNF{alpha} in Liver of ob/ob Mice Treated with PTP1B ASO
Because the decrease in p38 phosphorylation correlated with the decrease in glucose and the decrease in PTP1B protein and because downstream substrates of p38 have implications in diabetes, we were particularly interested in the consequences of increased basal p38 phosphorylation in ob/ob mice and the reversal of this state by PTP1B ASO. To this end, we investigated the status of substrates of p38 in liver of lean, ob/+ mice, and in liver of ob/ob mice treated with and without PTP1B ASO. p38 regulates the expression of TNF{alpha} at the level of translation (12, 27), and increased levels of TNF{alpha} have been associated with insulin resistance (28, 30, 38). To assess the effect of PTP1B reduction on the level of TNF{alpha} protein in liver, ELISAs for TNF{alpha} protein were performed on liver extracts. TNF{alpha} protein levels were decreased in the liver of ob/ob mice treated with 25 mg/kg PTP1B ASO (Fig. 3AGo). Microarray experiments comparing liver RNA from the 25 mg/kg PTP1B ASO ob/ob mice to the saline control ob/ob mice showed no decrease in TNF{alpha} RNA (Table 1Go), implying that the decrease may be posttranscriptional. [While the fold change ranged from a 1.3- to 6-fold increase, the Affymetrix program (Affymetrix, Inc., Santa Clara, CA) did not consider these changes to be statistically significant and thus called it as no change]. Real-time PCR experiments confirmed the lack of a decrease in the RNA level of TNF{alpha} and actually showed a statistically significant increase in TNF{alpha} RNA in the PTP1B ASO-treated ob/ob mice relative to the saline control-treated mice (Fig. 3BGo). These results further support the posttranscriptional nature of the decrease in TNF{alpha} protein in the 25 mg/kg PTP1B ASO-treated mice relative to the saline control-treated mice. The decrease in TNF{alpha} thus paralleled the decrease in p38 MAPK basal activity for the high-dose PTP1B ASO treatment and likely occurred in a posttranscriptional manner that is consistent with the regulation of TNF{alpha} by p38.



View larger version (38K):
[in this window]
[in a new window]
 
Figure 3. TNF{alpha} Protein, CREB Phosphorylation, and PEPCK RNA Levels Are Decreased in Liver of ob/ob Mice Treated with PTP1B ASO, whereas TNF{alpha} RNA Levels Are Increased

A, Quantitation of TNF{alpha} protein levels in liver using an ELISA. Real-time PCR results using RNA isolated from liver of saline control- and 25 mg/kg PTP1B ASO-treated ob/ob mice are shown for TNF{alpha} (B) and PEPCK (D). Western blotting and quantitation of phospho-CREB using liver extracts are shown (C) with the blot of phospho-protein on top and the whole protein blot below. For all panels, quantitation is shown relative to the ob/ob saline control group and represents the average ± SEM. Statistical analysis was performed using a two-tailed Student’s t test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

 

View this table:
[in this window]
[in a new window]
 
Table 1. Microarray Data Using Liver RNA from ob/ob Mice Treated with ASO-25 Relative to Saline Control for Selected Genes

 
The transcription factor CREB is a protein that can be regulated by p38 via phosphorylation (12, 14, 25, 26). Western blotting for phosphorylated CREB revealed an increase in CREB phosphorylation in ob/ob vs. lean, ob/+ mice. This high basal activity was decreased in the liver of ob/ob mice treated with PTP1B ASO at 25 mg/kg (Fig. 3CGo). The decrease in CREB phosphorylation paralleled the decrease in p38 phosphorylation and the decrease in plasma glucose particularly at the high dose of PTP1B ASO. Western blotting revealed no change in CREB protein levels (Fig. 3CGo, lower gel), and microarray experiments with the same liver samples concurred, indicating no change in CREB RNA (Table 1Go). Only the phosphorylation state of CREB was changed—consistent with the observed decrease in phosphorylation of p38. CREB regulates the transcription of PEPCK, a rate-limiting step in gluconeogenesis. Correlating with the decrease in CREB phosphorylation, RNA levels of PEPCK were decreased in liver of 25 mg/kg PTP1B ASO-treated ob/ob mice relative to saline control-treated mice as determined by both microarray (Table 1Go) and real-time PCR (Fig. 3DGo).

The Mechanism for Decreased p38 Phosphorylation in PTP1B ASO-Treated ob/ob Mice and in Lean, ob/+ Littermates Is Not through Decreased Phosphorylation of MKK3 or MKK6
The question remained as to the mechanism by which p38 phosphorylation was being decreased by PTP1B ASO or, looking from another perspective, how the basal activation state of p38 was being increased in diabetic ob/ob mice. Basal tyrosine phosphorylation of the IR was decreased in liver of mice treated with 25 mg/kg PTP1B ASO for 6 wk (Fig. 4AGo). This corresponded to the decrease in p38 phosphorylation but did not indicate if these events were directly connected from the IR to p38 via upstream kinases. Phosphorylation of MKK3 and MKK6, the upstream kinases of p38, was not increased in ob/ob vs. lean, ob/+ mice, and the ob/ob mice did not exhibit a decrease in MKK3/MKK6 phosphorylation at any dose of PTP1B ASO (Fig. 4BGo). MKK4 phosphorylation also remained unchanged in PTP1B ASO-treated animals and thus was not affecting p38 phosphorylation (data not shown). Therefore, the pathway leading to decreased phosphorylation of p38 MAPK was not direct from the IR via MKK3, MKK4, or MKK6. A previous study reported that p38 could be activated by protein kinase C (PKC) in vascular smooth muscle cells independent of MKK3 and MKK6 (36). Total PKC activation remained unchanged, however, in PTP1B ASO-treated mice as well as in lean littermates relative to saline control-treated ob/ob mice (data not shown).



View larger version (25K):
[in this window]
[in a new window]
 
Figure 4. Tyrosine Phosphorylation of the IR Is Decreased in Liver of ob/ob Mice Treated with PTP1B ASO; However, Phosphorylation of MKK3 and MKK6 Is Unaffected

Western blots and quantitation for phospho-Tyr (Y)-IR (A) and phospho-MKK3/6 (B) using liver extracts from the indicated groups. The top blot in each panel is the phospho-specific antibody blot, and the lower blot is the whole protein blot for the same protein. Quantitation, below the blots, shows changes in the phosphorylated protein relative to the ob/ob saline control group and represent the average ± SEM. Statistical analysis was performed using a two-tailed Student’s t test (***, P < 0.001).

 
p38 Is Not Activated in PTP1B ASO-Treated ob/ob Mice in Response to Insulin, Supporting the Lack of a Direct Response from the IR to p38
Further evidence that PTP1B ASO is not affecting p38 through a direct pathway involving the IR was derived from animals stimulated with insulin. A subset of vehicle control and 25 mg/kg PTP1B ASO-treated ob/ob mice were administered 2 U of insulin ip for 1 min or 5 min before animals were killed. Despite decreased basal tyrosine phosphorylation of the IR (Fig. 4AGo), PTP1B ASO-treated mice exhibited increased tyrosine phosphorylation of the IR, IRS-1, and IRS-2 and increased serine/threonine phosphorylation of protein kinase B (PKB) upon insulin stimulation (1 or 5 min) relative to vehicle control-treated PTP1B ASO mice and insulin-stimulated saline-control mice (8, 9). However, p38 phosphorylation was not significantly increased upon insulin stimulation for 1 min (Fig. 5Go). Activation with insulin for 5 min produced similar results (data not shown). Thus, increased tyrosine phosphorylation of the IR upon insulin stimulation did not produce a downstream increase in p38 phosphorylation. Phosphorylation of ERK was also not increased upon insulin stimulation (data not shown). This indicates a divergence of direct effects of the IR relating to metabolic vs. stress and mitogenic cascades in the PTP1B ASO-treated mice.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 5. Insulin Treatment Does Not Increase Activation of p38 in Liver of PTP1B ASO-Treated ob/ob Mice

Western blotting for phospho-p38 using liver extracts from saline-control or PTP1B ASO-treated (25 mg/kg, twice weekly for 6 wk) ob/ob mice challenged with an ip bolus of vehicle (-) or insulin (+) for 1 min before the animals were killed. The top blot is the phospho-specific antibody blot, whereas the lower blot is a whole protein blot for the same protein. Quantitation, below the blots, shows changes in the phosphorylated protein relative to the vehicle-treated saline control group (-). The data represent the average ± SEM. *, P < 0.05 using a two-tailed Student’s t test.

 
Decreased Basal Activity of p38 May Be Due at Least in Part to Increased Expression of MAPK Phosphatases
One possible mechanism by which the activation state of p38 may be decreased independently of the IR and MKK3/6 is through increased levels of MKPs (35, 39, 40, 41). Microarray experiments indicated increased mRNA levels of several homologs of MKPs (Table 1Go). Homologs of mouse MKP1 (also called CL100) and mouse PAC1, were increased in 25 mg/kg PTP1B ASO-treated ob/ob mouse liver relative to saline control-treated ob/ob mouse liver. These results support a potential role for phosphatases in reducing the level of p38 and ERK activation. To verify these results and more specifically identify which phosphatase might be increased in ASO-treated mice, Western blotting for PAC1 and MKP1 was performed on liver extracts from 25 mg/kg PTP1B ASO- and saline control-treated ob/ob mice. PAC1 (Fig. 6AGo) but not MKP1 (Fig. 6BGo) protein levels were increased in 25 mg/kg PTP1B ASO-treated mice relative to saline control-treated mice indicating that PAC1, which specifically dephosphorylates p38 and ERK (35, 39, 40, 41), may be involved in the decreased phosphorylation of these kinases in the PTP1B ASO-treated ob/ob mice.



View larger version (15K):
[in this window]
[in a new window]
 
Figure 6. PAC1 Protein Levels But Not MKP-1 Protein Levels Are Increased in PTP1B ASO- vs. Saline Control-Treated ob/ob Mice

Western blots of PAC1 (A) and MKP-1 (B) protein levels in liver extracts from saline control- and 25 mg/kg PTP1B ASO-treated ob/ob mice are shown. Quantitation, below the blots, shows changes relative to saline control-treated ob/ob mice. The data are expressed as average ± SEM (***, P < 0.001 using a two-tailed Student’s t test).

 
A Decrease in the Basal Activation State of p38 in Liver of ob/ob Mice Is Also Observed with Another Insulin Sensitizer, Rosiglitazone
To determine if the decrease in p38 phosphorylation is commonly associated with normalization of plasma glucose and increased insulin sensitivity or if the decrease was specifically related to PTP1B inhibition, the activation state of p38 was also examined in ob/ob mice treated with rosiglitazone. Treatment of ob/ob mice with rosiglitazone for 7 d resulted in normalization of plasma glucose (Fig. 7AGo) as reported previously (42). In addition, the basal activation state of p38 (Fig. 7BGo) was decreased in liver of rosiglitazone-treated ob/ob mice. This was true despite the different time course of study and the different mechanism of action for rosiglitazone relative to PTP1B ASO. Rosiglitazone works through a PPAR{alpha}/{gamma}-dependent mechanism (43, 44), whereas PTP1B ASO, instead, reduced PTP1B protein and had no effect on PPAR {alpha}/{gamma} RNA or PPAR{alpha}/{gamma} transcriptional targets in liver and actually decreased PPAR{gamma} RNA in fat of ob/ob mice (2, 45). Thus, two different insulin sensitizers—PTP1B ASO and rosiglitazone—reduce p38 phosphorylation in ob/ob mice and revert the diabetic phenotype of these mice.



View larger version (20K):
[in this window]
[in a new window]
 
Figure 7. Phosphorylation of p38 Is Also Decreased in Liver of ob/ob Mice Treated with Rosiglitazone

Western blots for plasma glucose (A) and phospho-p38 (B) using liver extracts from untreated ob/ob mice or mice treated with vehicle control or rosiglitazone at 10 mg/kg for 7 d. The top blot in panel B is the phospho-specific antibody blot, whereas the lower blot is a whole protein blot for the same protein. Quantitation, below the blots, shows changes in the phosphorylated protein relative to the ob/ob vehicle control group. The data represent the average ± SEM. ***, P < 0.001 using a two-tailed Student’s t test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Active p38 stimulates immune responses and other inflammatory pathways, and increased or constitutive activation of p38 can have adverse implications (12, 14, 27). In this study, we found that ob/ob mice exhibit increased hepatic p38 phosphorylation, and to a lesser extent ERK MAPK phosphorylation, relative to lean, ob/+ littermates. Treatment with PTP1B ASO decreased this constitutive basal activity to levels at or below lean littermate levels, which may ameliorate or prevent the negative consequences of constitutive activation and could help to diminish some of the inflammatory processes observed in diabetes (46). Further, p38 does not appear to be more sensitive to insulin stimulation in the ASO-treated ob/ob animals, despite the fact that other pathways such as the IR to IRS-1 and -2 to PKB pathway are more sensitive to insulin in these mice. These results suggest an improved biochemical phenotype with PTP1B ASO treatment.

Two substrates of p38, TNF{alpha} and CREB, were also affected in PTP1B ASO-treated mice. TNF{alpha} protein levels were decreased as was phosphorylation of CREB. Increased levels of TNF{alpha} have been linked as a risk factor for insulin resistance and diabetes (28, 29, 30). Because p38 regulates TNF{alpha} at the level of translation (27) and because the down-regulation of TNF{alpha} expression in the 25 mg/kg PTP1B ASO-treated mice was likely posttranscriptional, decreased activation of p38 may be responsible for the observed decrease in TNF{alpha} protein. However, the decreased activity of p38 may not be the only factor involved because lean, ob/+ littermates exhibited decreased basal p38 activity but exhibited only a small decrease in TNF{alpha} relative to ob/ob mice. Lean, ob/+ mice are somewhat insulin resistant (47), so other factors may affect TNF{alpha} levels in these animals. Alternatively, these results may simply be a reflection of the differing sensitivities of the assays. Nevertheless, the observed results demonstrating a lack of effect on MKK3 and MKK6 phosphorylation (the means by which TNF{alpha} affects p38 activity) suggest that reduction of p38 phosphorylation precedes TNF{alpha} reduction in these animals.

Previous reports have shown that increased levels of TNF{alpha} induce insulin resistance in fa/fa rats via increased expression of leukocyte antigen-related tyrosine phosphatase (LAR) resulting in decreased focal adhesion kinase (FAK) phosphorylation (38). Decreased TNF{alpha} expression in liver of these animals was also linked to decreased expression of LAR and increased phosphorylation of FAK resulting in increased insulin sensitivity (38). Because we observed decreased protein levels of TNF{alpha} in liver of the PTP1B ASO-treated ob/ob mice and because this correlated with increased insulin sensitivity in these animals, we investigated the effect on LAR expression and FAK phosphorylation. We observed no effect on LAR RNA or protein and no change in phosphorylation of FAK in these animals (data not shown). These results indicate that a decrease in TNF{alpha} is not sufficient to decrease LAR and that decreased levels of LAR are not required for increased sensitivity to insulin.

Activated p38 can phosphorylate the transcription factor CREB via downstream kinases (12, 14, 26). Here we observed CREB to be phosphorylated in liver of ob/ob mice relative to lean, ob/+ littermates. Decreased phosphorylation of CREB correlated with decreased phosphorylation of p38 in high-dose PTP1B ASO-treated ob/ob mice and saline-treated lean, ob/+ mice (for phospho-CREB relative to phospho-p38, r = 0.73 for saline-treated ob/ob, PTP1B ASO-25-treated ob/ob and saline-treated lean ob/+ mice). The decrease in CREB phosphorylation was not via PKB or PKC—two other kinases known to phosphorylate CREB (31, 34, 48, 49)—because PKB phosphorylation was increased in liver of PTP1B ASO high-dose animals and PKC activity remained unchanged (data not shown). There was, however, a 20% decrease in protein kinase A (PKA) activity (data not shown)—another kinase that can affect CREB phosphorylation (33)—in high dose PTP1B ASO-treated animals that may have contributed in part to the decrease in CREB phosphorylation. Interestingly, PKA was increased in the low dose PTP1B ASO-treated animals. Taken together, these results suggest there could be a cumulative effect of p38 activity and PKA activity on CREB phosphorylation. The possible interaction of PKA and p38 on CREB activation as well as subsequent effects on PEPCK transcription could help to explain the difference in glucose levels in the low dose vs. high dose PTP1B ASO-treated animals.

PEPCK is the rate-limiting step in gluconeogenesis, and its basal expression is regulated by phosphorylated CREB (31, 32, 33, 34). Expression of PEPCK RNA, as detected by microarray analysis and real-time PCR, was decreased in high dose PTP1B ASO-treated ob/ob mice relative to saline control-treated mice. The decrease in PEPCK RNA may be due at least in part to the decrease in phosphorylated CREB. In addition, decreased CREB phosphorylation in the ASO-treated mice correlated with the lowered plasma glucose levels observed in these mice (r = 0.61). These results from diabetic mice contradict an earlier report on diabetic rats that indicated a decrease in CREB phosphorylation and no correlation with PEPCK expression (50).

The lack of effect on MKK3 and MKK6, the upstream kinases of p38, was unexpected. The only reported evidence for activation of p38 independent of MKK3 and MKK6 was through PKC (36). We could detect no changes in PKC activity; however, it is possible that minor changes in one isoform could have been missed in the context of total PKC activity. Another indication that decreased phosphorylation of p38 was not likely a direct effect via the IR is that p38 did not show a significant increase in activation in response to insulin stimulation despite an increase in activation of the IR, IRS-2, and PKB in liver (8, 9). Based on our results, a plausible explanation is that increased expression of MKPs, or other similar signals, prevented the activation of these MAPK by insulin.

The mechanism for decreased phosphorylation of p38 in the liver of PTP1B ASO-treated ob/ob diabetic animals may thus be due at least in part to increased levels of MKPs. Microarray experiments revealed increased expression of expressed sequence tag (EST) homologs of MKPs in liver of PTP1B ASO-treated ob/ob mice relative to saline control-treated mice. Whereas the Affymetrix array listed these as being homologous to MKP1, the ESTs shared homology with other MKPs. The first and second MKP ESTs listed in Table 1Go were most homologous to mouse PAC1 [also called dual specificity phosphatase 2 (DSP2)] and next most homologous to mouse MKP1 (also called DSP1 and CL100) using standard basic local alignment search tool (BLAST) analysis (www.ncbi.nlm.nih.gov) but also showed homology to human and rat DSP7, DSP6 (also called MKP3), DSP9 (also called MKP4), DSP2 (PAC1), and MKP1 (35, 39, 40, 41). The third EST was most homologous to mouse MKP1 but also shared homology with mouse PAC1 as well as human and rat MKP1, PAC1, and DSP5. Although we cannot rule out a role for DSP6, DSP7, and DSP9 (antibodies to these phosphatases are not commercially available), Western blotting for PAC1 and MKP1 revealed that only PAC1 was increased in liver of PTP1B ASO-treated ob/ob mice relative to saline control-treated ob/ob mice. Because PAC1 is most specific for dephosphorylating p38 and ERK, whereas MKP1 can dephosphorylate ERK, p38 and JNK (35, 41), this result correlates well with our data showing a decrease in phosphorylation of p38 and ERK but no change in phosphorylation of JNK in PTP1B ASO-treated ob/ob mice. Taken together, these results suggest a role of increased levels of MKPs, especially PAC1, in the decreased activation of p38 and ERK in the PTP1B ASO-treated animals.

The mechanism by which PAC1 expression may have been increased in response to PTP1B ASO remains unclear. There have been reports that activation of phosphatidylinositol 3-kinase (PI-3K) can increase MKP expression via inducible nitric oxide synthase in vascular smooth muscle cells and that high glucose and insulin levels can inhibit MKP expression in these cells (51, 52, 53). Other reports have indicated that PI 3-K activity is decreased in liver of ob/ob mice relative to lean littermates (54). Whereas the total basal activity of PI 3-K has not been determined in the high dose PTP1B ASO-treated mice, hepatic IRS-2 associated PI 3-K activity was increased in these mice in the presence (5-fold) and absence (2-fold) of insulin (8). In addition, phosphorylation of PKB, which lies downstream of PI 3-K, was increased in liver of these mice both in the presence and absence of insulin stimulation (8). Thus, increased PI 3-K activity could be playing a role in increasing the level of MKP expression.

The data demonstrating a reduction in glucose and a reduction in phosphorylation of p38 in rosiglitazone-treated ob/ob mice raise interesting observations and possible hypotheses. These results show a correlation between decreased p38 phosphorylation and lowering of plasma glucose using two different agents (rosiglitazone and PTP1B ASO) that act through two different mechanisms. This raises the question of whether there may be a common connection between decreasing the phosphorylation of p38 and normalization of plasma glucose and even whether p38 activity may play a role in hyperglycemia. Although the causality and order of activation remain to be determined in future experiments, the decrease in p38 phosphorylation at the lower dose of PTP1B ASO, before an effect on glucose or insulin, would argue against this being simply an effect of decreased hyperosmolarity, hyperinsulinemia, or hyperglycemia on p38 activity. This is also supported by the lack of an effect on MKK3 and MKK6, the means by which high osmolarity, high insulin, or high glucose levels would be expected to affect p38 (12, 14, 23, 24). Because rosiglitazone has been reported to affect IRS-2 protein levels and PI 3-K activity (55, 56), PI 3-K activation and possible downstream effects on MKPs could be a potential link between PTP1B ASO and rosiglitazone in the lowering of p38. The effects of PTP1B ASO on signaling pathways and their possible relationships are shown in Fig. 8Go.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 8. Simplified Signaling Schematic Summarizing IR and MAPK Signal Transduction Pathways and How These Pathways Were Affected by Inhibition of PTP1B using PTP1B ASO

Thin arrows indicate activation directions; blunted black arrows indicate inhibition directions. Dark, thick arrows indicate basal effects after 6 wk of ASO treatment. Proteins that were activated in ASO-treated mice upon insulin stimulation are indicated by an asterisk. Dark arrows pointing up indicate an increase, dark arrows pointing down indicate a decrease relative to ob/ob saline controls. P, Phosphorylated protein effect.

 
In conclusion, we have found that inhibition of PTP1B, using an ASO, decreased constitutive phosphorylation of p38 and its substrate CREB in liver of ob/ob diabetic mice. This inhibition was consistent with reduction of gluconeogenesis as evidenced by decreased transcription of PEPCK and normalization of plasma glucose. In addition, production of TNF{alpha} was reduced through a posttranscriptional mechanism potentially involving decreased phosphorylation of p38. Finally, decreased phosphorylation of p38 was independent of MKK3 and MKK6 and appears to involve a novel mechanism that may be due at least in part to increased expression of PAC1. These studies support PTP1B as a plausible target for treatment of diabetes. In addition, our studies, particularly in conjunction with reports of increased p38 basal activity in aorta (36) and glomeruli (37) of diabetic rats, provide evidence for a role of increased activation of MAPKs in insulin resistance and diabetes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animal Studies
All animal experimentation was conducted in accord with accepted standards of humane animal care including Abbott Laboratories Institutional Animal Care and Use Committee guidelines. Detailed methods of the PTP1B ASO animal studies have been described previously (9, 45). Briefly, obese, diabetic C57Bl/6J ob/ob mice and their lean littermates (6–7 wk of age, The Jackson Laboratory, Bar Harbor, ME) were weighed and tail snip plasma glucose levels were determined by the glucose oxidase method (Precision G glucose meter, Abbott Laboratories, North Chicago, IL). Animals were randomized based on glucose levels and body weight, and baseline plasma insulin samples were measured (ELISA, ALPCO Diagnostics, Windham, NH). Two separate studies were performed. In the first study, treatment groups were: ob/ob mice treated with saline, a control oligonucleotide or PTP1B ASO at 50 mg/kg for 3 wk. In the second study, treatment groups were: ob/ob mice treated with saline (vehicle control) or PTP1B ASO at 25 mg/kg, 2.5 mg/kg, or 0.25 mg/kg (n = 10/treatment); and lean, ob/+ littermates treated with saline (n = 10/treatment) for 6 wk. All mice were dosed twice weekly ip. The PTP1B ASO (ISIS-113715) was identified and provided by Isis Pharmaceuticals (Carlsbad, CA); its identification has been described elsewhere (9, 45). Before use, PTP1B ASO was weighed and resuspended in saline at a concentration of 25 mg/ml and OD read at 260. The stock was diluted to the desired concentration for injection in sterile saline. At the end of each week, tail bleed glucose and insulin levels were determined under nonfasting conditions. At the end of 6 wk of treatment, animals were killed. Relevant tissues including liver, fat, and muscle were flash-frozen in liquid nitrogen and stored at -80 C. Before the animals were killed, satellite saline and ASO-treated (25 mg/kg) animals were administered saline or insulin after a 5-h fast. Insulin (or saline) was given ip at 2 U/kg in 0.1% BSA. Tissue samples from liver and fat (1 min and 5 min post treatment) were taken under both saline- and insulin-stimulated conditions then frozen and stored as indicated above.

In a separate experiment, ob/ob mice (6–7 wk of age, The Jackson Laboratory) were randomized as above using glucose measurements. Treatment groups were: untreated, vehicle-treated (hydroxypropyl methylcellulose), and rosiglitazone-treated (10 mg/kg·d) with n = 8 mice per group. All mice were dosed orally once daily for 7 d. Tail bleed glucose and insulin levels were determined on d 0 (before first dose), 1, 3, and 7 before dosing. Mice were killed 4 h after the last dose, and then liver, fat, and muscle were collected, flash frozen in liquid nitrogen, and stored at -80 C.

Tissue Extraction and RNA Isolation
RNA was prepared by grinding approximately 100 mg of liver tissue in 1 ml of TRIzol reagent. Analysis was performed according to the Affymetrix protocol. Briefly, for microarray analysis, the RNA from four mice in PTP1B ASO-treated or control groups was pooled using equal amounts to make a total of 20 µg of RNA. cRNA was prepared using the Superscript Choice system (Life Technologies, Inc., Rockville, MD). The protocol was followed with the exception that the primer used for the reverse transcription reaction was a modified T7 primer with 24 thymidines at the 5' end. The sequence was: 5'GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(deoxythymidine)24-3'. After this, labeled cRNA was synthesized according to the manufacturers instructions from the cDNA using the Enzo RNA Transcript Labeling Kit (Life Technologies, Inc.). Approximately 20 µg of cRNA were then fragmented in a solution of 40 mM Tris-acetate (pH 8.1), 100 mM KOAc, and 30 mM MgOAc at 94 C for 35 min.

Microarray Analysis and Real-Time PCR
Labeled cRNA was hybridized to the Affymetrix GeneChip Test2 Array (Affymetrix, Inc.) to verify the quality of labeled cRNA. After this, liver cRNA was hybridized to the Affymetrix MU11K A and B chip for saline control and 25 mg/kg PTP1B ASO ob/ob samples. The cRNA was hybridized overnight at 45 C. The data were analyzed using Affymetrix GeneChip version 3.2 software and Spotfire.Net version 5.0 (Spotfire, Inc., Cambridge, MA). To confirm the results of the microarray analysis for TNF{alpha} and PEPCK, real-time PCR was performed using the Taqman EZ RT-PCR Core Reagents kit (Perkin-Elmer Corp., part of Roche Molecular Biochemicals, Indianapolis, IN). For the analysis, 100 ng of total RNA was used. The reactions were done in triplicate on four individual mice per treatment group and the real time PCR experiment was performed twice. Two different PEPCK primer sets from different regions of the gene were used to determine the change in PEPCK RNA because the decrease in PEPCK as detected by microarray analysis was relatively small.

Protein Lysates and Protein Assays
One milliliter of lysis buffer [20 mM Tris-HCl (pH 7.4), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM EDTA, 25 mM ß-glycerophosphate, 20 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10 µg/ml leupeptin, 1 mM benzamidine, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrocholoride, 1 mM microcystin] was added to 50 mg of liver. Tissue was sonicated, inverted at 4 C for at least 30 min, and then pelleted at 14,000 rpm. Supernatants were split into two portions. One portion was mixed with sodium dodecyl sulfate loading buffer and used for gel electrophoresis and Western blotting. The second portion was assayed for protein content according to the method of Bradford (57) using protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA). Absorbance was read on a Spectramax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA) at a wavelength of 595 nm.

Western Blots, ELISAs, and Activity Assays
For Western blotting, 20 or 50 µg of protein extract (depending on the protein of interest) were subjected to SDS-PAGE using Criterion gels (Bio-Rad Laboratories, Inc.), then transferred to nitrocellulose. The nitrocellulose blots were incubated with a primary rabbit antibody to PTP1B (Upstate Biotechnology, Inc., Lake Placid, NY), phospho-p38, phospho-ERK, phospho-JNK, phospho-MKK3/6, phospho-MAPK/ERK kinase 1/2, phospho-MKK4, phospho-PKB, or phospho-CREB (Cell Signaling Technology, Beverly, MA) at dilutions of 1:1000 or with primary mouse antibody to phospho-tyrosine (PY99), MKP-1 or PAC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at dilutions of 1:2000, 1:500, and 1:400, respectively. This was followed by incubation with secondary antibody, donkey antirabbit or sheep antimouse (Amersham Pharmacia Biotech, Piscataway, NJ), or donkey antigoat (Santa Cruz Biotechnology, Inc.) at dilutions of 1:5000. Protein bands were detected by ECL (Amersham Pharmacia Biotech) as recommended by the manufacturer. Phospho-blots were stripped and reprobed with whole protein antibodies including ERK1/2, JNK1/2, p38, MKK3, MKK6, phospho-PKB, CREB (Santa Cruz Biotechnology, Inc.) or IR (PharMingen, part of BD Biosciences, San Diego, CA). Blots were quantitated on a densitometer (Molecular Dynamics, Inc., part of Amersham Pharmacia Biotech, Piscataway, NJ). Western blots were repeated two to four times each with at least two separate preparations of sample extracts. Four mice were analyzed per treatment group. TNF{alpha} was quantitated from liver by ELISA using a kit as recommended by the manufacturer (Biosource International, Camarillo, CA). The ELISA was repeated four times with fresh extracts each time. PKC and PKA activity was measured using a kit (Pierce Chemical Co., Rockford, IL) as recommended by the manufacturer. The assays were repeated two or three times with fresh extracts each time.

Statistical Analysis
Statistical analysis for all studies was performed with Excel (Microsoft Corp., Redmond, WA) analysis tools using a two-tailed Student’s t test with significance shown as *,P < 0.05; **, P < 0.01; ***, P < 0.001.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Brett Monia and Dr. Mandy Butler of ISIS Pharmaceuticals for helpful discussions during the course of the project. We also thank Dr. Christine Collins for her critical evaluation of and suggestions for the manuscript.


    FOOTNOTES
 
1 Present address: Pfizer Global R&D, La Jolla Laboratories, 10770 Science Center Drive, San Diego, California 92121-1187. Back

2 Present address: Rosetta Inpharmatics, 12040 115th Avenue, Kirkland, Washington 98034. Back

Abbreviations: ASO, Antisense oligonucleotide; CREB, cAMP response element binding protein; DSP, dual specificity phosphatase; EST, expressed sequence tag; FAK, focal adhesion kinase; IR, insulin receptor; IRS, IR substrate; JNK, Jun amino terminal kinase; LAR, leukocyte antigen-related tyrosine phosphatase; MKK, MAPK kinase; MKP, MAPK phosphatase; PAC1, phosphatase of activated cells; PEPCK, phosphoenolpyruvate carboxykinase; PI 3-K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PTP1B, protein tyrosine phosphatase 1B.

Received for publication August 19, 2002. Accepted for publication March 12, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Reaven GM 1988 Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607[Abstract]
  2. Kenner K, Anyanwu E, Olefsky J, Kusari J 1996 Protein-tyrosine phosphatase 1B is a negative regulator of insulin- and insulin-like growth factor-I-stimulated signaling. J Biol Chem 271:19810–19816[Abstract/Free Full Text]
  3. Seely B, Staubs P, Reichart D, Berhanu P, Milarski K, Saltiel A, Kusari J, Olefsky J 1996 Protein tyrosine phosphatase 1B interacts with the activated insulin receptor. Diabetes 45:1379–1385[Abstract]
  4. Calera M, Vallega G, Pilch P 2000 Dynamics of protein-tyrosine phosphatases in rat adipocytes. J Biol Chem 275:6308–6312[Abstract/Free Full Text]
  5. Ahmad F, Azevedo JJ, Cortright R, Dohm G, Goldstein B 1997 Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes. J Clin Invest 100:449–458[Abstract/Free Full Text]
  6. Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy A, Normandin D, Cheng A, Himms-Hagen J, Chan C-C, Ramachandran C, Gresser M, Tremblay M, Kennedy BP 1999 Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283:1544–1548[Abstract/Free Full Text]
  7. Klaman L, Boss O, Peroni O, Kim J, Martino J, Zabolotny J, Moghal N, Lubkin M, Kim Y, Sharpe A, Stricker-Krongrad A, Shulman G, Neel B, Kahn B 2000 Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 20:5479–5489[Abstract/Free Full Text]
  8. Gum RJ, Gaede LL, Koterski SL, Heindel M, Clampit JE, Zinker BA, Trevillyan JM, Ulrich RG, Jirousek MR, Rondinone CM 2003 Reduction of protein tyrosine phosphatase 1B increases insulin-dependent signaling in ob/ob mice. Diabetes 52:21–28[Abstract/Free Full Text]
  9. Zinker BA, Rondinone CM, Trevillyan JM, Gum RJ, Clampit JE, Waring JF, Xie N, Wilcox D, Jacobson P, Frost L, Kroeger PE, Reilly RM, Koterski S, Opgenorth TJ, Ulrich RG, Crosby S, Butler M, Murray SF, McKay RA, Bhanot S, Monia BP, Jirousek MR 2002 PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice. Proc Natl Acad Sci USA 99:11357–11362[Abstract/Free Full Text]
  10. Schmidt C, McKillop I, Cahill P, Sitzmann J 1997 Increased MAPK expression and activity in primary human hepatocellular carcinoma. Biochem Biophys Res Commun 236:54–58[CrossRef][Medline]
  11. Ito Y, Sasaki Y, Horimoto M, Wada S, Tanaka Y, Kasahara A, Ueki T, Hirano T, Yamamoto H, Fujimoto J, Okamoto E, Hayashi N, Hori M 1998 Activation of mitogen-activated protein kinases/extracellular signal-regulated kinases in hepatocellular human carcinoma. Hepatology 27:951–958[Medline]
  12. Kyriakis J, Avruch J 2001 Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807–869[Abstract/Free Full Text]
  13. Marshall C 1994 MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 4:82–89[Medline]
  14. Gum R, Young P 2001 p38 Inhibition. In: Ciliberto G, Savino R, eds. Cytokine inhibitors. New York: Marcel Dekker, Inc.; 329–361
  15. Cobb M, Goldsmith E 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
  16. Han J, Lee J, Bibbs L, Ulevitch R 1994 A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265:808–811[Medline]
  17. Lee J, Laydon J, McDonnell P, Gallagher T, Kumar S, Green D, McNulty D, Blumenthal M, Heys J, Landvatter S, Strickler J, McLaughlin M, Siemens I, Fisher S, Livi G, White J, Adams J, Young P 1994 A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:42–52
  18. Kumar S, McDonnell P, Gum R, Hand A, Lee J, Young P 1997 Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun 235:533–538[CrossRef][Medline]
  19. Derijard G, Hibi M, Wu I-H, Barrett T, Su B, Deng T, Karin M, Davis R 1994 JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025–1037[Medline]
  20. Raingeaud J, Gupta S, Rogers J, Dickens M, Han J, Ulevitch R, Davis R 1995 Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270:7420–7426[Abstract/Free Full Text]
  21. Davis R 1995 Transcriptional regulation of MAPK kinases. Mol Reprod Dev 42:459–467[Medline]
  22. Lazar D, Russell J, Brady M, Mastick C, Waters S, Yamauchi K, Pessin J, Cuatrecasas P, Saltiel A 1995 Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem 270:20801–20807[Abstract/Free Full Text]
  23. Avruch J 1998 Insulin signal transduction through protein kinase cascades. Mol Cell Biochem 182:31–48[CrossRef][Medline]
  24. Igarashi M, Yamaguchi H, Hirata A, Daimon M, Tominaga M, Kato T 2000 Insulin activates p38 mitogen-activated protein (MAP) kinase via a MAP kinase kinase (MKK) 3/MKK 6 pathway in vascular smooth muscle cells. Eur J Clin Invest 30:668–677[CrossRef][Medline]
  25. Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ 1996 FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. EMBO J 15:4629–4642[Abstract]
  26. Deak M, Clifton AD, Lucocq LM, Alessi DR 1998 Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J 17:4426–4441[Abstract/Free Full Text]
  27. Lee J, Young P 1996 Role of CSBP/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J Leuk Biol 59:152–157[Abstract]
  28. Hotamisligil G 1999 The role of TNF{alpha} and TNF receptors in obesity and insulin resistance. J Intern Med 425:621–625[CrossRef]
  29. Cheung A, Ree D, Kolls J, Fuselier J, Coy D, Bryer-Ash M 1998 An in vivo model for elucidation of the mechanism of tumor necrosis factor-{alpha} (TNF-{alpha})-induced insulin resistance: evidence for differential regulation of insulin signaling by TNF-{alpha}. Endocrinology 139:4928–4935[Abstract/Free Full Text]
  30. Lang C, Dobrescu C, Bagby G 1992 Tumor necrosis factor impairs insulin action on peripheral glucose disposal and hepatic glucose output. Endocrinology 130:43–52[Abstract]
  31. Andrisani OM 1999 CREB-mediated transcriptional control. Crit Rev Eukaryot Gene Expr 9:19–32[Medline]
  32. Xing L, Quinn PG 1993 Involvement of 3',5'-cyclic adenosine monophosphate regulatory element binding protein (CREB) in both basal and hormone-mediated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Mol Endocrinol 7:1484–1494[Abstract]
  33. Quinn PG 1994 Inhibition by insulin of protein kinase A-induced transcription of the phosphoenolpyruvate carboxykinase gene. Mediation by the activation domain of cAMP response element-binding protein (CREB) and factors bound to the TATA box. J Biol Chem 269:14375–14378[Abstract/Free Full Text]
  34. Mitchell J, Noisin E, Hall R, O’Brien R, Imai E, Granner D 1994 Integration of multiple signals through a complex hormone response unit in the phosphoenolpyruvate carboxykinase gene promoter. Mol Endocrinol 8:585–594[Abstract]
  35. Chu Y, Solski PA, Khosravi-Far R, der CJ, Kelly K 1996 The mitogen-activated protein kinase phosphatases PAC1, MKP-1 and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J Biol Chem 271:6497–6501[Abstract/Free Full Text]
  36. Masahiko I, Wakasaki H, Takahara N, Ishii H, Jiang Z-Y, Yamauchi T, Kuboki K, Meier M, Rhodes C, King G 1999 Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest 103:185–195[Abstract/Free Full Text]
  37. Kang S, Adler S, Lapage J, Natarajan R 2001 p38 MAPK and MAPK kinase 3/6 mRNA and activities are increased in early diabetic glomeruli. Kidney Int 60:543–552[CrossRef][Medline]
  38. Cheung A, Wang J, Ree D, Kolls J, Bryer-Ash M 2000 Tumor necrosis factor-{alpha} induces hepatic insulin resistance in obese zucker (fa/fa) rats via interaction of leukocyte antigen-related tyrosine phosphatase with focal adhesion kinase. Diabetes 49:810–819[Abstract]
  39. Keyse SM 1998 Protein phosphatases and the regulation of MAP kinase activity. Semin Cell Dev Biol 9:143–152[CrossRef][Medline]
  40. Martell KJ, Angelotti T, Ullrich A 1998 The "VH1-like" dual-specificity protein tyrosine phosphatases. Mol Cell 8:2–11[CrossRef]
  41. Camps M, Nichols A, Arkinstall S 1999 Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J 14:6–16
  42. Edvardsson U, Bergstrom M, Alexandersson M, Bamberg K, Ljung B, Dahllof B 1999 Rosiglitazone (BRL49653), a PPAR{gamma}-selective agonist, causes peroxisome proliferator-like liver effects in obese mice. J Lipid Res 40:1177–1184[Abstract/Free Full Text]
  43. Lebovitz H, Banerji M 2001 Insulin resistance and its treatment by thiazolidinediones. Recent Prog Horm Res 56:265–294[Abstract/Free Full Text]
  44. Greene D 1999 Rosiglitazone: a new therapy for type 2 diabetes. Expert Opin Investig Drugs 8:1709–1719[Medline]
  45. Rondinone CM, Trevillyan JM, Clampit J, Gum RJ, Berg C, Kroeger P, Frost L, Zinker BA, Reilly R, Ulrich R, Butler M, Monia B, Jirousek MR, Waring JF 2002 PTP-1B reduction regulates adiposity and expression of genes involved in lipogenesis. Diabetes 51:2405–2411[Abstract/Free Full Text]
  46. Pickup J, Crook M 1998 Is type II diabetes mellitus a disease of the innate immune system? Diabetologia 41:1241–1248[CrossRef][Medline]
  47. Flatt P, Bailey C 1981 Abnormal plasma glucose and insulin repsonses in heterozygous lean (ob/+) mice. Diabetologia 20:573–577[Medline]
  48. Agati JM, Yeagley D, Quinn PG 1998 Assessment of the roles of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase B and protein kinase C in insulin inhibition of cAMP-induced phosphoenolpyruvate carboxykinase gene transcription. J Biol Chem 723:18751–18759[CrossRef]
  49. Liao J, Barthel A, Nakatani K, Roth RA 1998 Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene. J Biol Chem 273:27320–27324[Abstract/Free Full Text]
  50. Davies G, Crosson S, Khandelwal F, Roesler W 1995 The phosphorylation state of the cAMP response element binding protein is decreased in diabetic rat liver. Arch Biochem Biophys 323:477–483[CrossRef][Medline]
  51. Begum N, Ragolia L, McCarthy M, Duddy N 1998 Regulation of mitogen-activated protein kinase phosphatase-1 induction by insulin in vascular smooth muscle cells. Evaluation of the role of the nitric oxide signaling pathway and potential defects in hypertension. J Biol Chem 273:25164–25170[Abstract/Free Full Text]
  52. Begum N, Ragolia L 2000 High glucose and insulin inhibit VSMC MKP-1 expression by blocking iNOS via p38 MAPK activation. Am J Physiol Cell Physiol 278:C81–C91
  53. Takehara N, Kawabe J, Aizawa Y, Hasebe N, Kikuchi K 2000 High glucose attenuates insulin-induced mitogen-activated protein kinase phosphatase-1 (MKP-1) expression in vascular smooth muscle cells. Biochem Biophys Acta 1497:244–252[CrossRef][Medline]
  54. Folli F, Saad M, Backer J, Kahn C 1993 Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus. J Clin Invest 92:1787–1794[Medline]
  55. Standaert M, Kanoh Y, Sajan M, Bandyopadhyay G, Farese R 2002 Cbl, IRS-1, and IRS-2 mediate effects of rosiglitazone on PI3K, PKC-{lambda}, and glucose transport in 3T3/L1 adipocytes. Endocrinology 143:1705–1716[Abstract/Free Full Text]
  56. Yang C, Chang TJ, Chang JC, Liu MW, Tai TY, Hsu WH, Chuang LM 2001 Rosiglitazone (BRL 49653) enhances insulin secretory response via phosphatidylinositol 3-kinase pathway. Diabetes 50:2598–2602[Abstract/Free Full Text]
  57. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]