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BACKGROUND AND PURPOSE Skeletal muscle dysfunction is a major comorbidity of chronic obstructive pulmonary disease (COPD). This type of muscle dysfunction may be a direct consequence of oxidative insults evoked by cigarette smoke (CS) exposure. The present study examined the effects of a potent Nox inhibitor and reactive oxygen species (ROS) scavenger, apocynin, on CS-induced muscle dysfunction. EXPERIMENTAL APPROACH Male BALB/c mice were exposed to either room air (sham) or CS generated from nine cigarettes per day, 5 days a week for 8 weeks, with or without the coadministration of apocynin (5 mg·kg-1 , i.p.). C2C12 myotubes exposed to either hydrogen peroxide (H2 O2 ) or water-soluble cigarette smoke extract (CSE) with or without apocynin (500 nM) were used as an experimental model in vitro. KEY RESULTS Eight weeks of CS exposure caused muscle dysfunction in mice, reflected by 10% loss of muscle mass and 54% loss of strength of tibialis anterior which were prevented by apocynin administration. In C2C12 myotubes, direct exposure to H2 O2 or CSE caused myofibre wasting, accompanied by ~50% loss of muscle-derived insulin-like growth factor (IGF)-1 and two-fold induction of Cybb, independent of cellular inflammation. Expression of myostatin and MAFbx, negative regulators of muscle mass, were up-regulated under H2 O2 but not CSE conditions. Apocynin treatment abolished CSE-induced Cybb expression, preserving muscle-derived IGF-1 expression and signalling pathway downstream of mammalian target of rapamycin (mTOR), thereby preventing myofibre wasting. CONCLUSION AND IMPLICATIONS Targeted pharmacological inhibition of Nox-derived ROS may alleviate the lung and systemic manifestations in smokers with COPD.

Background and purpose It is well established that both smokers and patients with COPD are at a significantly heightened risk of cardiovascular disease (CVD), although the mechanisms underpinning the onset and progression of co-morbid CVD are largely unknown. Here, we explored whether cigarette smoke (CS) exposure impairs vascular function in mice and given the well-known pathological role for oxidative stress in COPD, whether the antioxidant compound ebselen prevents CS-induced vascular dysfunction in mice. Experimental approach Male BALB/c mice were exposed to either room air (sham) or CS generated from nine cigarettes per day, 5 days a week for 8 weeks. Mice were treated with ebselen (10 mg·kg-1 , oral gavage once daily) or vehicle (5% w/v CM cellulose in water) 1 h prior to the first CS exposure of the day. Upon killing, bronchoalveolar lavage fluid (BALF) was collected to assess pulmonary inflammation, and the thoracic aorta was excised to investigate vascular endothelial and smooth muscle dilator responses ex vivo. Key results CS exposure caused a significant increase in lung inflammation which was reduced by ebselen. CS also caused significant endothelial dysfunction in the thoracic aorta which was attributed to a down-regulation of eNOS expression and increased vascular oxidative stress. Ebselen abolished the aortic endothelial dysfunction seen in CS-exposed mice by reducing the oxidative burden and preserving eNOS expression. Conclusion and implications Targeting CS-induced oxidative stress with ebselen may provide a novel means for treating the life-threatening pulmonary and cardiovascular manifestations associated with cigarette smoking and COPD.

Background and ObjectiveNeurocognitive dysfunction is present in up to ∼61% of people with chronic obstructive pulmonary disease (COPD), with symptoms including learning and memory deficiencies, negatively impacting the quality of life of these individuals. As the mechanisms responsible for neurocognitive deficits in COPD remain unknown, we explored whether chronic cigarette smoke (CS) exposure causes neurocognitive dysfunction in mice and whether this is associated with neuroinflammation and an altered neuropathology.MethodsMale BALB/c mice were exposed to room air (sham) or CS (9 cigarettes/day, 5 days/week) for 24 weeks. After 23 weeks, mice underwent neurocognitive tests to assess working and spatial memory retention. At 24 weeks, mice were culled and lungs were collected and assessed for hallmark features of COPD. Serum was assessed for systemic inflammation and the hippocampus was collected for neuroinflammatory and structural analysis.ResultsChronic CS exposure impaired lung function as well as driving pulmonary inflammation, emphysema, and systemic inflammation. CS exposure impaired working memory retention, which was associated with a suppression in hippocampal microglial number, however, these microglia displayed a more activated morphology. CS-exposed mice showed changes in astrocyte density as well as a reduction in synaptophysin and dendritic spines in the hippocampus.ConclusionWe have developed an experimental model of COPD in mice that recapitulates the hallmark features of the human disease. The altered microglial/astrocytic profiles and alterations in the neuropathology within the hippocampus may explain the neurocognitive dysfunction observed during COPD.

Gastrointestinal (GI) dysfunction is a common comorbidity of chronic obstructive pulmonary disease (COPD) for which a major cause is cigarette smoking (CS). The underlying mechanisms and precise effects of CS on gut contractility, however, are not fully characterised. Therefore, the aim of the present study was to investigate whether CS impacts GI function and structure in a mouse model of CS-induced COPD. We also aimed to investigate GI function in the presence of ebselen, an antioxidant that has shown beneficial effects on lung inflammation resulting from CS exposure. Mice were exposed to CS for 2 or 6 months. GI structure was analysed by histology and immunofluorescence. After 2 months of CS exposure, ex vivo gut motility was analysed using video-imaging techniques to examine changes in colonic migrating motor complexes (CMMCs). CS decreased colon length in mice. Mice exposed to CS for 2 months had a higher frequency of CMMCs and a reduced resting colonic diameter but no change in enteric neuron numbers. Ten days cessation after 2 months CS reversed CMMC frequency changes but not the reduced colonic diameter phenotype. Ebselen treatment reversed the CS-induced reduction in colonic diameter. After 6 months CS, the number of myenteric nitric-oxide producing neurons was significantly reduced. This is the first evidence of colonic dysmotility in a mouse model of CS-induced COPD. Dysmotility after 2 months CS is not due to altered neuron numbers; however, prolonged CS-exposure significantly reduced enteric neuron numbers in mice. Further research is needed to assess potential therapeutic applications of ebselen in GI dysfunction in COPD.

Skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease negatively impacts quality of life and survival. Cigarette smoking (CS) is the major risk factor for chronic obstructive pulmonary disease and skeletal muscle dysfunction; however, how CS affects skeletal muscle function remains enigmatic. To examine the impact of CS on skeletal muscle inflammation and regeneration, male BALB/c mice were exposed to CS for 8 weeks before muscle injury was induced by barium chloride injection, and were maintained on the CS protocol for up to 21 days after injury. Barium chloride injection resulted in architectural damage to the tibialis anterior muscle, resulting in a decrease contractile function, which was worsened by CS exposure. CS exposure caused muscle atrophy (reduction in gross weight and myofiber cross-sectional area) and altered fiber type composition (31% reduction of oxidative fibers). Both contractile function and loss in myofiber cross-sectional area by CS exposure gradually recovered over time. Satellite cells are muscle stem cells that confer skeletal muscle the plasticity to adapt to changing demands. CS exposure blunted Pax7+ centralized nuclei within satellite cells and thus prevented the activation of these muscle stem cells. Finally, CS triggered muscle inflammation; in particular, there was an exacerbated recruitment of F4/80+ monocytic cells to the site of injury along with enhanced proinflammatory cytokine expression. In conclusion, CS exposure amplified the local inflammatory response at the site of skeletal muscle injury, and this was associated with impaired satellite cell activation, leading to a worsened muscle injury and contractile function without detectable impacts on the recovery outcomes.

Background Cigarette smoking (CS) is the leading cause of chronic obstructive pulmonary disease (COPD). The “spill-over” of pulmonary inflammation into the systemic circulation may damage the brain, leading to cognitive dysfunction. Cessation of CS can improve pulmonary and neurocognitive outcomes, however, its benefit on the neuroinflammatory profile remains uncertain. Here, we investigate how CS exposure impairs neurocognition and whether this can be reversed with CS cessation or an antioxidant treatment. Methods Male BALB/c mice were exposed to CS (9 cigarettes/day for 8 weeks) followed by 4 weeks of CS cessation. Another cohort of CS-exposed mice were co-administrated with a glutathione peroxidase mimetic, ebselen (10 mg/kg) or vehicle (5% CM-cellulose). We assessed pulmonary inflammation, spatial and working memory, and the hippocampal microglial, oxidative and synaptic profiles. Results CS exposure increased lung inflammation which was reduced following CS cessation. CS caused spatial and working memory impairments which were attributed to hippocampal microglial activation and suppression of synaptophysin. CS cessation did not improve memory deficits or alter microglial activation. Ebselen completely prevented the CS-induced working and spatial memory impairments, which was associated with restored synaptophysin expression without altering microglial activation. Conclusion We were able to model the CS-induced memory impairment and microglial activation seen in human COPD. The preventative effects of ebselen on memory impairment is likely to be dependent on a preserved synaptogenic profile. Cessation alone also appears to be insufficient in correcting the memory impairment, suggesting the importance of incorporating antioxidant therapy to help maximising the benefit of cessation.

Consumption of diet rich in fat and cigarette smoking (CS) are independent risk factors of non-alcoholic steatohepatitis (NASH), and they often occur together in some populations. The present study investigated the mechanisms of high-fat diet (HFD) and CS, individually and in combination, on the pathogenesis of NASH in mice. C57BL/6 male mice were subjected to either a low-fat chow (CH) or HFD with or without mainstream CS-exposure (4 cigarettes/day, 5 days/ week for 14 weeks). HFD alone caused hepatosteatosis (2.5-fold increase in TG content) and a significant increase in 3-nitrotyrisine (by ∼40-fold) but without an indication of liver injury, inflammation or fibrosis. CS alone in CH-fed mice increased in Tnfα expression and macrophage infiltration by 2-fold and relatively less increase in 3-nitrotyrosine (18-fold). Combination of HFD and CS precipitated hepatosteatosis to NASH reflected by exacerbated makers of liver inflammation and fibrosis which were associated with much severe liver oxidative stress (90-fold increase in 3-nitrotyrisine along with 6-fold increase in carbonylated proteins and 56% increase in lipid oxidations). Further studies were performed to administer the antioxidant tempol to CS exposed HFD mice and the results showed that the inhibition of liver oxidative stress prevented inflammatory and fibrotic changes in liver despite persisting hepatosteatosis. Our findings suggest that oxidative stress is a key mechanism underlying CS-promoted progression of simple hepatosteatosis to NASH. Targeting hepatic oxidative stress may be a viable strategy in halting the progression of metabolic associated fatty liver disease.

Chronic obstructive pulmonary disease (COPD) is a major incurable global health burden and is currently the 4th largest cause of death in the world. Importantly, much of the disease burden and health care utilisation in COPD is associated with the management of its comorbidities (e.g. skeletal muscle wasting, ischemic heart disease, cognitive dysfunction) and infective viral and bacterial acute exacerbations (AECOPD). Current pharmacological treatments for COPD are relatively ineffective and the development of effective therapies has been severely hampered by the lack of understanding of the mechanisms and mediators underlying COPD. Since comorbidities have a tremendous impact on the prognosis and severity of COPD, the 2015 American Thoracic Society/European Respiratory Society (ATS/ERS) Research Statement on COPD urgently called for studies to elucidate the pathobiological mechanisms linking COPD to its comorbidities. It is now emerging that up to 50% of COPD patients have metabolic syndrome (MetS) as a comorbidity. It is currently not clear whether metabolic syndrome is an independent co-existing condition or a direct consequence of the progressive lung pathology in COPD patients. As MetS has important clinical implications on COPD outcomes, identification of disease mechanisms linking COPD to MetS is the key to effective therapy. In this comprehensive review, we discuss the potential mechanisms linking MetS to COPD and hence plausible therapeutic strategies to treat this debilitating comorbidity of COPD.

Background and Purpose: Cigarette smoking (CS) is the major risk factor for developing COPD and related skeletal muscle dysfunction. It has been postulated that CS exposure may directly causes muscle dysfunction via the induction of oxidative stress. The present study examined the effect of a potent Nox inhibitor and ROS scavenger, apocynin on CS-induced muscle dysfunction. Experimental Approach: Male BALB/c mice were exposed to either room air (sham) or CS generated from 9 cigarettes per day, 5 days a week for 8 weeks with or without apocynin treatment (5 mg·kg-1 w/v, intraperitoneal injection). C2C12 myotubes exposed to either hydrogen peroxide (H2O2) or water-soluble cigarette smoke extract (CSE) with or without apocynin (500 nM), was set up as an experimental model in vitro. Key Results: Eight weeks of CS exposure caused significant lung inflammation and muscle dysfunction in mice; evidenced by a 10% loss in muscle mass and 54% loss in contractile function of tibialis anterior, attributable to altered myogenic homeostasis and protein oxidation. These effects were prevented by apocynin administration. In C2C12 myotubes, direct exposure to H2O2 or CSE caused myofiber wasting, which was associated with altered myogenic homeostasis marked by ~50% loss in muscle-derived insulin-like growth factor (IGF)-1 and 1.5-fold increase in myostatin expression. Apocynin treatment completely attenuated CSE-induced Nox2 expression, preserving muscle-derived IGF-1 expression and downstream mammalian target of rapamycin (mTOR) signaling pathway, thereby preventing myofiber wasting. Conclusion and Implications: Targeted pharmacological inhibition of Nox-derived ROS may alleviate the lung and systemic manifestations in smokers with COPD.

Cigarette smoking (CS) is known to reduce body weight and this often masks its real effect on insulin action. The present study tested the hypothesis that CS can divert lipid deposition to muscles to offset the supposed benefit of reduced body weight gain on insulin signalling in this major site for glucose tolerance (or insulin action). The study was conducted in mice exposed to chronic CS followed by either a chow (CH) diet or a high-fat (HF) diet. CS increased triglyceride (TG) levels in both plasma and muscle despite a reduced body weight gain and adiposity. CS led to glucose intolerance in CH-fed mice and they retained the glucose intolerance that was induced by the HF diet. In adipose tissue, CS increased macrophage infiltration and the mRNA expression of TNFα but suppressed the protein expression of adipose triglyceride lipase and PPARγ. While CS increased hormone-sensitive lipase and suppressed the mRNA expression of leptin, these effects were blunted in HF-fed mice. These results imply that CS impairs insulin signalling in skeletal muscle via accumulated intramuscular lipids from lipolysis and lipodystrophy of adipose tissues. This may explain why smokers may not benefit from insulin sensitising effects of reduced body weight gain.

Endoplasmic reticulum (ER) stress has been implicated in the development of hypertension 3 through the induction of endothelial impairment. As 3′,4′-dihydroxyflavonol (DiOHF) 4 reduces vascular injury caused by ischaemia/reperfusion or diabetes, and flavonols have been demonstrated to attenuate ER stress, we investigated whether DiOHF can protect mice from ER stress-induced endothelial dysfunction. Male C57BLK/6 J mice were injected with tunicamycin to induce ER stress in the presence or absence of either DiOHF or tauroursodeoxycholic acid (TUDCA), an inhibitor of ER stress. Tunicamycin elevated blood pressure and impaired endothelium-dependent relaxation. Moreover, in aortae there was evidence of ER stress, oxidative stress and reduced NO production. This was coincident with increased NOX2 expression and reduced phosphorylation of endothelial nitric oxide synthase (eNOS) on Ser1176. Importantly, the effects of tunicamycin were significantly ameliorated by DiOHF or TUDCA. DiOHF also inhibited tunicamycin-induced ER stress and apoptosis in cultured human endothelial cells (HUVEC). These results provide evidence that ER stress is likely an important initiator of endothelial dysfunction through the induction of oxidative stress and a reduction in NO synthesis and that DiOHF directly protects against ER stress- induced injury. DiOHF may be useful to prevent ER and oxidative stress to preserve endothelial function, for example in hypertension.

The metabolic syndrome is associated with an increase in the activation of the renin angiotensin system, whose inhibition reduces the incidence of new-onset diabetes. Importantly, angiotensin II (AngII), independently of its vasoconstrictor action, causes b-cell inflammation and dysfunction, which may be an early step in the development of type 2 diabetes. The aim of this study was to determine how AngII causes b-cell dysfunction. Islets of Langerhans were isolated from C57BL/6J mice that had been infused with AngII in the presence or absence of taurineconjugated ursodeoxycholic acid (TUDCA) and effects on endoplasmic reticulum (ER) stress, inflammation, and b-cell function determined. The mechanism of action of AngII was further investigated using isolated murine islets and clonal b cells. We show that AngII triggers ER stress, an increase in the messenger RNA expression of proinflammatory cytokines, and promotes b-cell dysfunction in murine islets of Langerhans both in vivo and ex vivo. These effects were significantly attenuated by TUDCA, an inhibitor of ER stress. We also show that AngII-induced ER stress is required for the increased expression of proinflammatory cytokines and is caused by reactive oxygen species and IP3 receptor activation. These data reveal that the induction of ER stress is critical for AngII-induced b-cell dysfunction and indicates how therapies that promote ER homeostasis may be beneficial in the prevention of type 2 diabetes. © 2017 Endocrine Society.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER) causes ER stress and activation of unfolded protein response (UPR). This response can trigger ER-associated degradation and autophagy, which clear unfolded proteins and restore protein homeostasis. Recently, it has become clear that ubiquitination plays an important role in the regulation of autophagy. In the present study, we investigated how the E3 ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 (Nedd4-2) interacts with ER stress and autophagy. In mice, we found that an increase in the expression of Nedd4-2, which was concomitant with the activation of the UPR and autophagy, was caused by a prolonged high-fructose and high-fat diet that induces ER stress in the liver. Pharmacologic induction of ER stress also led to an increase in Nedd4-2 expression in cultured cells, which was coincident with UPR and autophagy activation. The inhibition of inositol-requiring enzyme 1 significantly suppressed Nedd4-2 expression. Moreover, increased Nedd4-2 expression in vivo was closely associated with the activation of inositol-requiring enzyme 1 and increased expression of the spliced form of X-box binding protein 1. Furthermore, knockdown of Nedd4-2 in cultured cells suppressed both basal autophagy and ER stress-induced autophagy, whereas overexpression of Nedd4-2-induced autophagy. Taken together, our findings provide evidence that Nedd4-2 is up-regulated in response to ER stress by the spliced form of X-box binding protein 1 and that this is important in the induction of an appropriate autophagic response.-Wang, H. Sun, R.-Q., Camera, D., Zeng, X.-Y., Jo, E., Chan, S. M. H., Herbert, T. P., Molero, J. C., Ye, J.-M. Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy.

Endoplasmic reticulum (ER) stress and the activation of the unfolded protein response (UPR) have been implicated in the pathogenesis of many common human diseases. Integral to the UPR and an important determinant in cell fate is the expression of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). This is promoted by activating transcription factor 4 (ATF4) whose expression is rapidly up-regulated in response to ER stress through an eIF2α phosphorylation-dependent increase in protein synthesis. Our data demonstrates that this ER stress-induced increase in ATF4 and CHOP expression is initiated by an increase in Atf4 and Chop mRNA, which is also dependent upon eIF2α phosphorylation. Despite being dependent on eIF2α phosphorylation, we provide evidence that these increases in Atf4 and Chop mRNA expression may occur independently of de novo protein synthesis. Moreover, we show that ER stress-induced Chop mRNA expression is exacerbated by Sirtuin-1 (SIRT1) inhibition indicating that changes in the energy status of the cell may play an important role in its regulation. This work highlights and extends previous findings, and provides important new insights into the mechanism of ER stress-induced expression of Atf4 and Chop mRNA that clearly warrants further investigation.

Endoplasmic reticulum (ER) stress is suggested to cause hepatic insulin resistance by increasing de novo lipogenesis (DNL) and directly interfering with insulin signaling through the activation of the c-Jun N-terminal kinase (JNK) and IκB kinase (IKK) pathway. The current study interrogated these two proposed mechanisms in a mouse model of hepatic insulin resistance induced by a high fructose (HFru) diet with the treatment of fenofibrate (FB) 100 mg/kg/day, a peroxisome proliferator–activated receptor α (PPARα) agonist known to reduce lipid accumulation while maintaining elevated DNL in the liver. FB administration completely corrected HFru-induced glucose intolerance, hepatic steatosis, and the impaired hepatic insulin signaling (pAkt and pGSK3β). Of note, both the IRE1/XBP1 and PERK/eIF2α arms of unfolded protein response (UPR) signaling were activated. While retaining the elevated DNL (indicated by the upregulation of SREBP1c, ACC, FAS, and SCD1 and [3H]H2O incorporation into lipids), FB treatment markedly increased fatty acid oxidation (indicated by induction of ACOX1, p-ACC, β-HAD activity, and [14C]palmitate oxidation) and eliminated the accumulation of diacylglycerols (DAGs), which is known to have an impact on insulin signaling. Despite the marked activation of UPR signaling, neither JNK nor IKK appeared to be activated. These findings suggest that lipid accumulation (mainly DAGs), rather than the activation of JNK or IKK, is pivotal for ER stress to cause hepatic insulin resistance. Therefore, by reducing the accumulation of deleterious lipids, activation of PPARα can ameliorate hepatic insulin resistance against increased ER stress.

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes.

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of κB kinase, and tumor necrosis factor-α, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro . Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Neonatal obesity predisposes individuals to obesity throughout life. In rats, neonatal overfeeding also leads to early accelerated weight gain that persists into adulthood. The phenotype is associated with dysfunction in a number of systems including paraventricular nucleus of the hypothalamus (PVN) responses to psychological and immune stressors. However, in many cases weight gain in neonatally overfed rats stabilizes in early adulthood so the animal does not become more obese as it ages. Here we examined if neonatal overfeeding by suckling rats in small litters predisposes them to exacerbated metabolic and central inflammatory disturbances if they are also given a high fat diet in later life. In adulthood we gave the rats normal chow, 3 days, or 3 weeks high fat diet (45% kcal from fat) and measured peripheral indices of metabolic disturbance. We also investigated hypothalamic microglial changes, as an index of central inflammation, as well as PVN responses to lipopolysaccharide (LPS). Surprisingly, neonatal overfeeding did not predispose rats to the metabolic effects of a high fat diet. Weight changes and glucose metabolism were unaffected by the early life experience. However, short term (3 day) high fat diet was associated with more microglia in the hypothalamus and a markedly exacerbated PVN response to LPS in control rats; effects not seen in the neonatally overfed. Our findings indicate neonatally overfed animals are not more susceptible to the adverse metabolic effects of a short-term high fat diet but may be less able to respond to the central effects.

The unfolded protein response (UPR) pathways have been implicated in the development of hepatic insulin resistance during high fructose (HFru) feeding. The present study investigated their roles in initiating impaired insulin signaling transduction in the liver induced by HFru feeding in mice. HFru feeding resulted in hepatic steatosis, increased de novo lipogenesis and activation of two arms of the UPR pathways (IRE1/XBP1 and PERK/eIF2α) in similar patterns from 3days to 8weeks. In order to identify the earliest trigger of impaired insulin signaling in the liver, we fed mice a HFru diet for one day and revealed that only the IRE1 branch was activated (by 2-fold) and insulin-mediated Akt phosphorylation was blunted (~25%) in the liver. There were significant increases in phosphorylation of JNK (~50%) and IRS at serine site (~50%), protein content of ACC and FAS (up to 2.5-fold) and triglyceride level (2-fold) in liver (but not in muscle or fat). Blocking IRE1 activity abolished increases in JNK activity, IRS serine phosphorylation and protected insulin-stimulated Akt phosphorylation without altering hepatic steatosis or PKCε activity, a key link between lipids and insulin resistance. Our findings together suggest that activation of IRE1–JNK pathway is a key linker of impaired hepatic insulin signaling transduction induced by HFru feeding.

This review discusses issues largely from the biological point of view about the targeted approaches for the use of natural products for the discovery of anti-diabetic drugs in collaboration with medicinal chemists and computer-aided drug design. A major thrust of this review reflects the collaborative research of four institutions: RMIT University (Australia), Garvan Institute of Medical Research (Australia), Shanghai Institute of Materia Medica of the Chinese Academy of Science (China) and Sun-Yat Sen University (China) in the past eight years. By joining forces of biomedical research in diabetes and medicinal chemistry with a focus on traditional medicine, they are trying to bridge the West (the latest research discoveries in biomedical research) with the East (traditional medicine) to step forward in drug discovery from natural products. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-beta (NF-kappabeta). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-kappabeta before exercise, but the nuclear abundance of p-p38 MAPK was higher (P

Reducing lipid accumulation in insulin target tissues is critical for the treatment of type 2 diabetes. This study aimed to develop a biochemical assay in cells for high throughput (HTP) screening of anti-diabetic drugs by reducing lipid accumulation via different mechanisms. We designed a new method to extract triglyceride (TG) with KOH to allow biochemical quantification of TGs for HTP screening in 3T3-L1 cells. This new method was validated for its biochemical properties with identical results of TG obtained with or without KOH (r(2) = 0.9978, p0.001) and a fourfold improvement in TG extraction recovery rate (88-95%, p0.001) as compared to the conventional chloroform/methanol extraction (12-18%). The ability of this phenotype screening to capture potential anti-diabetic drugs was verified by pharmacological agents well known to alter lipid accumulation by different mechanisms including AMPK activators, fatty acid synthesis inhibitors, PPARγ activator and several lipogenic substrates. To further demonstrate the application of this screening tool for discovery of new anti-diabetic drugs, we screened200 new candidates selected from Chinese medicine and identified 49 compounds from different classes which reduced TG content by50% at 1 μM or75% at 10 μM. Finally, we tested two selected leads (albiflorin and oxymatrine) in vivo and confirmed their efficacy in reducing visceral adiposity, glucose intolerance and hepatic steatosis in high fat-fed or high fructose-fed mice. Our results indicate that screening for the efficacy on lipid accumulation in cells by biochemical quantification of TGs with KOH extraction is an effective tool for the identification of new anti-diabetic compounds.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK α2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK α2 activity reduced the association of AMPK with glycogen and increased AMPK α2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at ~70% VO2 peak on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK α2 activity and acetyl-CoA carboxylase β phosphorylation, increased translocation of AMPK α2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK α2 under conditions of low muscle glycogen enhances AMPK α2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle.Key words: exercise, subcellular localization, glycogen binding domain, AMP-activated protein kinase.

To determine the expression and induction of cytokines in human skeletal muscle during concentric contractions, eight males performed 60 min of bicycle exercise, with either a normal (Con) or reduced (Lo Gly) preexercise intramuscular glycogen content. Muscle biopsy samples were obtained before and after exercise and analyzed for glycogen and the mRNA expression of 13 cytokines. Resting muscle glycogen was higher ( P < 0.05) in Con compared with Lo Gly and was reduced ( P < 0.05) to 102 ± 32 vs. 17 ± 5 mmol U glycosyl/kg dry mass for Con and Lo Gly, respectively. We detected mRNA levels in human skeletal muscle for five cytokines, namely interleukin (IL)-1β, IL-6, IL-8, IL-15, and tumor necrosis factor-α. However, muscle contraction increased ( P < 0.05) the mRNA expression of IL-6 and IL-8 alone. In addition, the fold change for both IL-8 and IL-6 was markedly higher ( P < 0.05) in Lo Gly compared with Con. Given these results, we analyzed venous blood samples, obtained before and during exercise, for IL-6 and IL-8. Plasma IL-6 was not different at rest, and although the circulating concentration of this cytokine increased ( P < 0.05) it increased to a greater extent ( P < 0.05) throughout exercise in Lo Gly. In contrast, plasma IL-8 was not affected by exercise or treatment. These data demonstrate that cytokines are not ubiquitously expressed in skeletal muscle and that only IL-6 and IL-8 mRNA are increased during contraction of this mode and duration. Furthermore, the mRNA abundance of IL-6 and IL-8 appears to be influenced by glycogen availability in the contracting muscle.

Hormone-sensitive lipase (HSL), an important regulatory enzyme for triacylglycerol hydrolysis within skeletal muscle, is controlled by beta-adrenergic signaling as well as intrinsic factors related to contraction and energy turnover. In the current study, we tested the capacity of 5'AMP-activated protein kinase (AMPK) to suppress beta-adrenergic stimulation of HSL activity. Eight male subjects completed 60 min of cycle exercise at 70% VO2 peak on two occasions: either with normal (CON) or low (LG) pre-exercise muscle glycogen content, which is known to enhance exercise-induced AMPK activity. Muscle samples were obtained before and immediately after exercise. Pre-exercise glycogen averaged 375 +/- 35 and 163 +/- 27 mmol x kg(-1) dm for CON and LG, respectively. AMPK alpha-2 was not different between trials at rest and was increased (3.7-fold, P

Hepatic steatosis is often associated with insulin resistance as a hallmark of the metabolic syndrome in the liver. The present study investigated the effects of PPARα activation induced by fenofibrate (FB) on the relationship of insulin resistance and hepatic steatosis in mice fed a high-fat (HF) diet, which increases lipid influx into the liver. Mice were fed HF diet to induce insulin resistance and hepatic steatosis with or without FB. FB activated PPARα and ameliorated HF diet-induced glucose intolerance and hepatic insulin resistance without altering either hepatic steatosis or inflammation signaling (JNK or IKK). Interestingly, FB treatment simultaneously increased fatty acid (FA) synthesis (50%) and oxidation (66%, both p