Kendra K. Bence

Liver-Specific Deletion of Protein-Tyrosine Phosphatase 1B (PTP1B) Improves Metabolic Syndrome and Attenuates Diet-Induced Endoplasmic Reticulum Stress

OBJECTIVE—The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling; consequently, mice deficient in PTP1B are hypersensitive to insulin. Because PTP1B−/− mice have diminished fat stores, the extent to which PTP1B directly regulates glucose homeostasis is unclear. Previously, we showed that brain-specific PTP1B−/− mice are protected against high-fat diet–induced obesity and glucose intolerance, whereas muscle-specific PTP1B−/− mice have increased insulin sensitivity independent of changes in adiposity. Here we studied the role of liver PTP1B in glucose homeostasis and lipid metabolism. RESEARCH DESIGN AND METHODS—We analyzed body mass/adiposity, insulin sensitivity, glucose tolerance, and lipid metabolism in liver-specific PTP1B−/− and PTP1Bfl/fl control mice, fed a chow or high-fat diet. RESULTS—Compared with normal littermates, liver-specific PTP1B−/− mice exhibit improved glucose homeostasis and lipid profiles, independent of changes in adiposity. Liver-specific PTP1B−/− mice have increased hepatic insulin signaling, decreased expression of gluconeogenic genes PEPCK and G-6-Pase, enhanced insulin-induced suppression of hepatic glucose production, and improved glucose tolerance. Liver-specific PTP1B−/− mice exhibit decreased triglyceride and cholesterol levels and diminished expression of lipogenic genes SREBPs, FAS, and ACC. Liver-specific PTP1B deletion also protects against high-fat diet–induced endoplasmic reticulum stress response in vivo, as evidenced by decreased phosphorylation of p38MAPK, JNK, PERK, and eIF2α and lower expression of the transcription factors C/EBP homologous protein and spliced X box-binding protein 1. CONCLUSIONS—Liver PTP1B plays an important role in glucose and lipid metabolism, independent of alterations in adiposity. Inhibition of PTP1B in peripheral tissues may be useful for the treatment of metabolic syndrome and reduction of cardiovascular risk in addition to diabetes.

Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation.

Medial nucleus tractus solitarius (mNTS) neurons express leptin receptors (LepRs), and intra-mNTS delivery of leptin reduces food intake and body weight. Here, the contribution of endogenous LepR signaling in mNTS neurons to energy balance control was examined. Knockdown of LepR in mNTS and area postrema (AP) neurons of rats (LepRKD) via adeno-associated virus short hairpin RNA-interference (AAV-shRNAi) resulted in significant hyperphagia for chow, high-fat, and sucrose diets, yielding increased body weight and adiposity. The chronic hyperphagia of mNTS/AP LepRKD rats is likely mediated by a reduction in leptin potentiation of gastrointestinal satiation signaling, as LepRKD rats showed decreased sensitivity to the intake-reducing effects of cholecystokinin. LepRKD rats showed increased basal AMP-kinase activity in mNTS/AP micropunches, and pharmacological data suggest that this increase provides a likely mechanism for their chronic hyperphagia. Overall these findings demonstrate that LepRs in mNTS and AP neurons are required for normal energy balance control.

PTP1B and SHP2 in POMC neurons reciprocally regulate energy balance in mice

Protein tyrosine phosphatase 1B (PTP1B) and SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) have been shown in mice to regulate metabolism via the central nervous system, but the specific neurons mediating these effects are unknown. Here, we have shown that proopiomelanocortin (POMC) neuron-specific deficiency in PTP1B or SHP2 in mice results in reciprocal effects on weight gain, adiposity, and energy balance induced by high-fat diet. Mice with POMC neuron-specific deletion of the gene encoding PTP1B (referred to herein as POMC-Ptp1b-/- mice) had reduced adiposity, improved leptin sensitivity, and increased energy expenditure compared with wild-type mice, whereas mice with POMC neuron-specific deletion of the gene encoding SHP2 (referred to herein as POMC-Shp2-/- mice) had elevated adiposity, decreased leptin sensitivity, and reduced energy expenditure. POMC-Ptp1b-/- mice showed substantially improved glucose homeostasis on a high-fat diet, and hyperinsulinemic-euglycemic clamp studies revealed that insulin sensitivity in these mice was improved on a standard chow diet in the absence of any weight difference. In contrast, POMC-Shp2-/- mice displayed impaired glucose tolerance only secondary to their increased weight gain. Interestingly, hypothalamic Pomc mRNA and alpha-melanocyte-stimulating hormone (alphaMSH) peptide levels were markedly reduced in POMC-Shp2-/- mice. These studies implicate PTP1B and SHP2 as important components of POMC neuron regulation of energy balance and point to what we believe to be a novel role for SHP2 in the normal function of the melanocortin system.

Improved Glucose Homeostasis in Mice with Muscle-Specific Deletion of Protein-Tyrosine Phosphatase 1B

ABSTRACT Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle triglycerides and serum adipokines are also affected similarly by HFD in both groups. Nevertheless, muscle-specific PTP1B−/− mice exhibit increased muscle glucose uptake, improved systemic insulin sensitivity, and enhanced glucose tolerance. These findings correlate with and are most likely caused by increased phosphorylation of the insulin receptor and its downstream signaling components. Thus, muscle PTP1B plays a major role in regulating insulin action and glucose homeostasis, independent of adiposity. In addition, rosiglitazone treatment of HFD-fed control and muscle-specific PTP1B−/− mice revealed that rosiglitazone acts additively with PTP1B deletion. Therefore, combining PTP1B inhibition with thiazolidinediones should be more effective than either alone for treating insulin-resistant states.

Intracellular Signals Mediating the Food Intake-Suppressive Effects of Hindbrain Glucagon-like Peptide-1 Receptor Activation

Glucagon-like peptide-1 receptor (GLP-1R) activation within the nucleus tractus solitarius (NTS) suppresses food intake and body weight (BW), but the intracellular signals mediating these effects are unknown. Here, hindbrain (fourth i.c.v.) GLP-1R activation by Exendin-4 (Ex-4) increased PKA and MAPK activity and decreased phosphorylation of AMPK in NTS. PKA and MAPK signaling contribute to food intake and BW suppression by Ex-4, as inhibitors RpcAMP and U0126 (fourth i.c.v.), respectively, attenuated Ex-4s effects. Hindbrain GLP-1R activation inhibited feeding by reducing meal number, not meal size. This effect was attenuated with stimulation of AMPK activity by AICAR (fourth i.c.v.). The PKA, MAPK, and AMPK signaling responses by Ex-4 were present in immortalized GLP-1R-expressing neurons (GT1-7). In conclusion, hindbrain GLP-1R activation suppresses food intake and BW through coordinated PKA-mediated suppression of AMPK and activation of MAPK. Pharmacotherapies targeting these signaling pathways, which mediate intake-suppressive effects of CNS GLP-1R activation, may prove efficacious in treating obesity.

Dorsal Hindbrain 5′-Adenosine Monophosphate-Activated Protein Kinase as an Intracellular Mediator of Energy Balance

The fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been implicated in central nervous system control of energy balance. Hypothalamic AMPK activity is increased by food deprivation, and this elevation is inhibited by refeeding or by leptin treatment. The contribution of extrahypothalamic AMPK activity in energy balance control has not been addressed. Here, we investigate the effects of physiological state on the AMPK activity in hindbrain nucleus tractus solitarius (NTS) neurons because treatments that reduce energy availability in these neurons trigger behavioral, endocrine, and autonomic responses to restore energy balance. Food-deprived rats showed significantly increased AMPK activity in both NTS- and hypothalamus-enriched lysates compared with those that were ad libitum fed. Pharmacological inhibition of AMPK activity in medial NTS neurons, by intraparenchymal injection of compound C, suppressed food intake and body weight gain compared with vehicle. Fourth ventricle (4th i.c.v.) compound C delivery increased heart rate and spontaneous activity in free-moving rats. Suppression of AMPK activity has been implicated in leptins anorectic action in the hypothalamus. Given the role of leptin signaling in food intake inhibition within the medial NTS, we also examined whether stimulation of hindbrain AMPK by 4th i.c.v. administration of 5-aminoimidazole-4-carboxamide-riboside (AICAR), an AMP-mimicking promoter of AMPK activity, could attenuate the inhibition of food intake by 4th i.c.v. leptin. The intake-suppressive effects of leptin (at 2 and 4 h) were completely reversed by AICAR. We conclude that 1) hindbrain AMPK activity contributes to energy balance control through regulation of food intake and energy expenditure, 2) leptins intake-reducing effects in the NTS are mediated by AMPK, and 3) central nervous system AMPK controls whole-body homeostasis at anatomically distributed sites across the neuraxis.

Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress

Obesity is associated with induction of the ER (endoplasmic reticulum)-stress response signalling and insulin resistance. PTP1B (protein tyrosine phosphatase 1B) is a major regulator of adiposity and insulin sensitivity. The aim of the present study was to investigate the role of L-PTP1B (liver-specific PTP1B) in chronically HFD (high-fat diet) and pharmacologically induced (tunicamycin and thapsigargin) ER-stress response signalling in vitro and in vivo. We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling. We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER stress, in vitro and in vivo. Silencing PTP1B in hepatic cell lines or mouse liver (L-PTP1B(-/-)) protected against induction of pharmacologically induced and/or obesity-induced ER stress. The HFD-induced increase in CHOP (CCAAT/enhancer-binding protein homologous protein) and BIP (binding immunoglobulin protein) mRNA levels were partially inhibited, whereas ATF4 (activated transcription factor 4), GADD34 (growth-arrest and DNA-damage-inducible protein 34), GRP94 (glucose-regulated protein 94), ERDJ4 (ER-localized DnaJ homologue) mRNAs and ATF6 protein cleavage were completely suppressed in L-PTP1B(-/-) mice relative to control littermates. L-PTP1B(-/-) mice also had increased nuclear translocation of spliced XBP-1 (X box-binding protein-1) via increased p85α binding. We demonstrate that the ER-stress response and L-PTP1B expression are interlinked in obesity- and pharmacologically induced ER stress and this may be one of the mechanisms behind improved insulin sensitivity and lower lipid accumulation in L-PTP1B(-/-) mice.

Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways.

Background— Heart failure is the leading cause of death in the United States. By delineating the pathways that regulate cardiomyocyte function, we can better understand the pathogenesis of cardiac disease. Many cardiomyocyte signaling pathways activate protein tyrosine kinases. However, the role of specific protein tyrosine phosphatases (PTPs) in these pathways is unknown. Methods and Results— Here, we show that mice with muscle-specific deletion of Ptpn11, the gene encoding the SH2 domain–containing PTP Shp2, rapidly develop a compensated dilated cardiomyopathy without an intervening hypertrophic phase, with signs of cardiac dysfunction appearing by the second postnatal month. Shp2-deficient primary cardiomyocytes are defective in extracellular signal–regulated kinase/mitogen-activated protein kinase (Erk/MAPK) activation in response to a variety of soluble agonists and pressure overload but show hyperactivation of the RhoA signaling pathway. Treatment of primary cardiomyocytes with Erk1/2- and RhoA pathway–specific inhibitors suggests that both abnormal Erk/MAPK and RhoA activities contribute to the dilated phenotype of Shp2-deficient hearts. Conclusions— Our results identify Shp2 as the first PTP with a critical role in adult cardiac function, indicate that in the absence of Shp2 cardiac hypertrophy does not occur in response to pressure overload, and demonstrate that the cardioprotective role of Shp2 is mediated via control of both the Erk/MAPK and RhoA signaling pathways.

Neuronal Protein Tyrosine Phosphatase 1B Deficiency Results in Inhibition of Hypothalamic AMPK and Isoform-Specific Activation of AMPK in Peripheral Tissues

ABSTRACT PTP1B−/− mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptins metabolic effects. We find that α1 and α2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B−/− mice. The effects of PTP1B deficiency on α2, but not α1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B−/− mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B−/− mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.

Adipocyte-Specific Protein Tyrosine Phosphatase 1B Deletion Increases Lipogenesis, Adipocyte Cell Size and Is a Minor Regulator of Glucose Homeostasis

Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B−/−) were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B−/− mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD)-fed adip-crePTP1B−/− mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR) and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α) expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.