Hepatic PTP1B Deficiency: The Promise of a Treatment for Metabolic Syndrome?

Abstract

Metabolic syndrome and type 2 diabetes are complex disorders that are associated with obesity, aging, and genetic predisposition. The increasing prevalence of metabolic abnormalities worldwide presents a serious public health problem, with rates of obesity and diabetes reaching unprecedented levels. A common feature of these disorders is the development of insulin resistance, resulting in decreased insulin-stimulated glucose uptake, failure to suppress hepatic glucose production, and accumulation of hepatic lipid. Recent studies in mice have shown that deficiency of the non-receptor protein tyrosine phosphatase, PTP1B, in liver leads to a host of improvements in metabolic parameters, including improved hepatic insulin sensitivity, reduced liver triglycerides, lower serum and hepatic cholesterol levels, and protection against high-fat diet-induced endoplasmic reticulum (ER) stress. Based on these promising studies, PTP1B inhibitors may prove to be a valuable therapeutic tool in the fight against metabolic syndrome and its associated comorbidities. In this review, the role of PTP1B in hepatic insulin sensitivity, hepatic lipid accumulation, and ER stress are discussed.

Metabolic syndrome and hepatic insulin resistance

Metabolic syndrome is characterized by a constellation of disorders, including obesity, dyslipidemia, impaired glucose tolerance, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD) (1). Many features of metabolic syndrome are linked to the development of insulin resistance, a condition that precedes the development of type 2 diabetes and often occurs well before diabetes is diagnosed in patients (2, 3). The liver plays a central role in the maintenance of normal glucose homeostasis and lipid metabolism, as demonstrated by the phenotype of mice with liver-specific deletion of the insulin receptor (LIRKO mice). LIRKO mice have “pure” hepatic insulin resistance resulting in incomplete suppression of hepatic glucose production and severe glucose intolerance (4). These mice also develop proatherogenic lipoprotein profiles, as demonstrated by increased VLDL cholesterol and decreased HDL cholesterol, and are more susceptible to cholesterol gallstone formation (4–6). Chronic inflammation associated with obesity is thought to induce insulin resistance in the liver via induction of inflammatory cytokines and adipokines which impair insulin signaling in hepatocytes (7).

A hallmark feature of metabolic syndrome is the accumulation of fat in the liver, which can progress from benign fatty liver to steatohepatitis and cirrhosis (8). Although the molecular mechanisms responsible for hepatic lipid accumulation are not well understood, it appears to be associated with the development of insulin resistance (7). Therefore, improvements in insulin resistance should have important therapeutic implications for the prevention and/or treatment of early stage fatty liver and associated diabetes risk. In fact, recent studies using insulin-sensitizing agents (thiazolidinediones and metformin) showed promising results in this regard (9). It should be noted, however, that a causal link between the development of hepatic insulin resistance and fatty liver has been difficult to prove; the study of genetically altered mouse models should prove useful in sorting out which features of metabolic syndrome are a direct result of impaired insulin signaling (1). Furthermore, although the detailed mechanism(s) underlying the development of insulin resistance remain controversial (10–13), there is general agreement that impaired post-IR signaling is involved (14).

Insulin signaling

Insulin is secreted from pancreatic β cells directly into the portal circulation; insulin promotes the storage of nutrients and is the major hormonal regulator of glucose homeostasis (4, 15). Insulin affects many components of hepatic carbohydrate and lipid metabolism, including glycogen synthesis, lipogenesis, and gluconeogenesis. These effects are achieved by modification of key molecular targets, either by reversible phosphorylation or by alteration of gene expression. In the liver insulin stimulates the expression glycolytic and lipogenic genes and suppresses genes encoding gluconeogenic enzymes (16).

Insulin action is mediated by insulin receptors (IRs), tyrosine kinases which reside on the plasma membranes of responsive cells (10, 14). Activation of the IR leads to transphosphorylation of tyrosine residues in the IR activation loop which, in turn, leads to enhanced ability of the IR to phosphorylate insulin receptor substrates (IRSs) (17). Tyrosyl phosphorylated IRS proteins act as docking sites for SH2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K). PI3K becomes activated upon binding to IRS proteins, and its phosphoinositide products facilitate the activation of downstream targets (10). Understanding how insulin signaling is dysregulated is very important since insulin resistance is linked to a host of metabolic abnormalities, including diabetes, hypertension, stroke, lipid abnormalities, cardiovascular disease, fatty liver disease, and infertility (18). Insulin signaling is terminated by dephosphorylation of key tyrosine residues on the insulin receptor and IRS proteins by protein tyrosine phosphatases (PTPs), including PTP1B (19–21).

PTP1B and insulin sensitivity

PTP1B is an abundant enzyme, tethered to the endoplasmic reticulum (ER) via its hydrophobic C-terminal targeting sequence (22, 23). Early studies of cultured cells, as well as structural studies, suggested that PTP1B might inhibit insulin signaling by dephosphorylating the IR and possibly IRS1 (24–32). Consistent with these in vitro studies, complete absence of PTP1B in mice (PTP1B^−/−) results in increased systemic insulin sensitivity, improved glucose tolerance, and enhanced muscle and liver IR phosphorylation, establishing PTP1B as a physiologically important IR phosphatase (33, 34). Furthermore, mice with neuronal-, muscle-, or liver-specific PTP1B-deficiency also display improved insulin sensitivity (35–38). PTP1B likely contributes to the pathogenesis of insulin resistance since it is over-expressed in many rodent and human models of obesity and insulin resistance (30, 39–45). In addition, transgenic over-expression of PTP1B in muscle or liver induces insulin resistance in mice (46, 47). Associations between PTP1B polymorphisms and insulin resistance, as well as aspects of metabolic syndrome in humans, also are reported (32, 48–58). Collectively, these studies have identified PTP1B as an attractive therapeutic target for obesity, diabetes, dyslipidemia, and metabolic syndrome (30, 31, 37, 59–62).

Liver-specific function of PTP1B

In light of the critical role of the liver in glucose homeostasis and the pathogenesis of metabolic syndrome, we asked whether PTP1B has autonomous effects on hepatic control of glucose homeostasis and lipid metabolism by generating liver-specific PTP1B^−/− mice (Albumin-Cre; Ptpn1^loxP/loxP). We found that disruption of PTP1B expression in the liver leads to increased hepatic insulin signaling, enhanced insulin suppression of hepatic glucose production, reduced serum and hepatic triglyceride and cholesterol levels, and protection against ER stress in mice fed a high-fat diet (HFD) (37). These metabolic parameters are improved despite no effect of hepatic PTP1B-deficiency on body weight or adiposity. Consistent with these results, liver-specific re-expression of PTP1B in total body PTP1B^−/− mice using heptaotropic adenovirus resulted in attenuation of insulin sensitivity coincident with decreased insulin-stimulated tyrosine phosphorylation of the IR in liver (46).

Other studies have also reported a link between PTP1B and liver-specific aspects of the metabolic syndrome. PTP1B protein levels are up-regulated in liver biopsies from patients with nonalcoholic steatohepatitis (63). Furthermore, PTP1B protein levels and activity are significantly higher in hepatocytes of fructose-fed hamsters, a model of insulin resistance and fatty liver disease (64). Hepatic PTP1B levels are also elevated in mice fed a high-fat diet (45). Furthermore, chronic fructose feeding resulted in elevated plasma VLDL levels in wild type mice, but not in PTP1B^−/− mice, suggesting an important role for PTP1B in regulating hepatic lipoprotein secretion (65). The factors responsible for increased PTP1B gene transcription in these rodent models is not clear, however recent studies have found that PTP1B expression can be induced by the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) in hepatocyte cell lines (45). In addition, glucose has been shown to enhance transcription of the PTP1B gene in a human hepatocyte cell line at least partially via PKC activity (66).

Hepatic ER stress

Insulin resistance may also develop as a result of obesity-induced ER stress response. Prolonged high-fat feeding in mice leads to increased ER stress response in liver, and the development of insulin resistance via serine phosphorylation of IRS1 (67). Hepatocytes contain abundant ER, which is a site of secretory and membrane protein synthesis and modification. Upon disruption in either protein folding or modification within the ER, the “unfolded protein response” (UPR) is activated (68, 69). The UPR deals with the adverse effects of ER stress and enhances cell survival via signaling through three stress-sensing proteins found on the ER membrane: PKR-like eukaryotic initiation factor 2α kinase (PERK), inositol-requiring kinase-1α (IRE-1α), and activating transcription factor-6 (ATF-6)(68, 69). The combined activation of these pathways results in upregulation of genes encoding ER-resident chaperones and proteins involved in degradation, and downregulation of genes involved in protein synthesis (70). Prolonged ER stress, however, leads to apoptosis, inflammation, and hepatic lipid accumulation (71). The importance of an intact UPR response is essential in peripheral tissues such as adipose and liver, which are susceptible to insulin resistance (72).

We found that mice with liver-specific PTP1B-deficiency are protected against high-fat diet-induced hepatic ER stress. Specifically, levels of p38 MAPK, JNK, PERK, and eIF2α phosphorylation were lower in Alb-Cre-PTP1B^−/− livers compared to wild type controls after prolonged high-fat diet exposure. Expression of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) and X-box binding protein (XBP-1) spliced isoform were also reduced in Alb-Cre-PTP1B^−/− livers, indicating these mice are more resistant to obesity-induced ER stress (37). Consistent with these studies, PTP1B^−/− primary and immortalized fibroblasts exhibit reduced ER stress response via impaired IRE-1 signaling (73). Protection against diet-induced ER stress could be a potential mechanism for the improved glucose and lipid homeostasis in Alb-Cre-PTP1B^−/− mice.

Insulin resistance and SREBP1

In addition to its effects on glucose homeostasis, insulin can alter hepatic lipogenic gene transcription. When circulating levels of insulin are elevated, sterol regulatory element binding protein 1c (SREBP1c) gene transcription is activated even after patients become “insulin resistant” at the level of gluconeogenesis (74), leading to increased lipogenic gene expression and hepatic triglyceride accumulation (75–77). LIRKO mice have low serum triglyceride levels and decreased SREBP1c gene expression (6), suggesting an important role for the IR in mediating these effects. It should be noted, however, that LIRKO mice have significantly elevated circulating leptin levels (78), and leptin has been suggested to play a role in regulation of glucose homeostasis (79) and plasma triglycerides (80). Thus, non-IR mediated pathways may be mediating the altered lipid profile in LIRKO mice (1).

Hepatic SREBP1c and SREBP1a gene expression levels were significantly lower in Alb-Cre-PTP1B^−/− mice than in wild type controls, which is counterintuitive to what would be expected from the LIRKO phenotype and the enhanced insulin sensitivity seen in Alb-Cre-PTP1B^−/−mice. Interestingly, treatment of leptin-deficient ob/ob mice with PTP1B oligonucleotides resulted in a similar decrease in lipogenic gene expression, including SREBP1 (81). Furthermore, rats fed a high fructose diet developed insulin resistance coincident with increased PTP1B and SREBP1 gene expression in the liver. Further investigation revealed that PTP1B may regulate SREBP1a and SREBP1c mRNA expression via phosphatase 2A (PP2A) activity (82). We therefore suspect that PTP1B may affect SREBP1 gene expression via a pathway distinct from the insulin signaling.

Hepatic leptin action

PTP1B is an important negative regulator of leptin signaling (83–85); mice with neuronal PTP1B-deficiency are lean due to enhanced hypothalamic leptin signaling and increased energy expenditure (35, 38). In addition to the central effects of PTP1B on leptin resistance, hepatic adenoviral overexpression of PTP1B in leptin-deficient ob/ob mice results in leptin resistance (86), and leptin has been shown to induce PTP1B expression in various tissues (87–89). Little is known about leptin action in the liver. Leptin has been shown to “signal” in the liver, however, and diet-induced obese rats have reduced hepatic levels of leptin receptor transcripts (90, 91). Furthermore, leptin treatment of wild type mice results in increased mRNA expression of several isoforms of the leptin receptor, including the long form of the receptor (ObRb)(92), suggesting the liver may be an important site of leptin action. However, mice with liver-specific ablation of the leptin receptor (ObR^Alb KO mice) are metabolically normal on a regular chow diet; phenotypes were not reported for mice fed a high-fat diet (93). Leptin can influence insulin signaling in the liver, although the relationship between hepatic leptin and insulin is complex. Leptin has been shown to stimulate glucose transport, but on the other hand has been shown to inhibit insulin-induced phosphorylation of IRS proteins which should lead to insulin resistance (94, 95). High circulating levels of leptin seen in obesity and metabolic syndrome may therefore contribute to hepatic steatosis indirectly by promoting insulin resistance (95). Liver-specific PTP1B^−/−mice might be expected to be leptin hypersensitive and thus be insulin resistant with fatty livers; however, the phenotypes of improved insulin signaling and lower hepatic fat accumulation in Alb-PTP1B^−/−mice suggest that PTP1B may not be regulating leptin signaling in hepatocytes. Alternatively, leptin may be signaling in the liver via a short form of the leptin receptor which does not utilize Jak2 (the direct PTP1B substrate which modulates leptin signaling in the hypothalamus), or the effects of hepatic PTP1B-deficiency on other pathways prevail in vivo.

Promise of PTP1B inhibitors in treating metabolic syndrome

The escalation in prevalence of metabolic syndrome and associated disorders highlights the urgent need for pharmacological treatments of these conditions. As a negative regulator of both insulin and leptin signaling, PTP1B represents an attractive target for the treatment insulin and leptin resistance (60, 61, 96–100). Based on the recent analysis of mice with liver-specific PTP1B-deficiency, PTP1B inhibitors may also be useful for the treatment of fatty liver disease. This notion is supported by a recent study showing that the dietary supplement curcumin inhibits PTP1B and prevents hepatic steatosis in fructose-fed rats (101). In addition, treatment of hyperglycemic IRS2^−/−mice with the antioxidant resveratrol resulted in decreased PTP1B mRNA and activity in the liver, resulting in improved insulin sensitivity (102). Although inhibiting central PTP1B activity may be required to produce effects on body weight (35, 38), studies in mice with peripheral PTP1B-deficiency, as well as anti-sense studies, indicate that peripherally acting inhibitors may also be useful for improving insulin sensitivity and dyslipidemia (36, 37, 81, 103, 104). More studies are needed in order to examine the tissue-specific effects of inhibiting PTP1B. Despite the potential promise of PTP1B inhibitors, the success of targeting this phosphatase has been limited due primarily to poor oral availability, and difficulty in inhibiting PTP1B without simultaneously inhibiting the closely related T-cell protein tyrosine phosphatase (TCPTP) (reviewed in (96, 98). These hurdles will have to be overcome to generate effective, specific PTP1B inhibitors for therapeutic use.