Stanley M.H. Chan

Differing Endoplasmic Reticulum Stress Response to Excess Lipogenesis versus Lipid Oversupply in Relation to Hepatic Steatosis and Insulin Resistance

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<0.01 vs Con). Glucose intolerance (iAUC increased by ∼60%) and blunted insulin-stimulated hepatic Akt and GSK3β phosphorylation (∼40–60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & β-HAD). As expected, DNL was increased (∼60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance.

Activation of PPARα ameliorates hepatic insulin resistance and steatosis in high fructose-fed mice despite increased endoplasmic reticulum stress.

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.

β-adrenergic stimulation of skeletal muscle HSL can be overridden by AMPK signaling

Hormone‐sensitive lipase (HSL), an important regulatory enzyme for triacylglycerol hydrolysis within skeletal muscle, is controlled by β‐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 β‐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•kg–1 dm for CON and LG, respectively. AMPK α‐2 was not different between trials at rest and was increased (3.7‐fold, P<0.05) by exercise during LG only. HSL activity did not differ between trials at rest and increased (0 min: 1.67 ± 0.13; 60 min: 2.60 ± 0.26 mmol•min–1•kg–1 dm) in CON. The exercise‐induced increase in HSL activity was attenuated by AMPK α‐2 activation in LG. The attenuated HSL activity during LG occurred despite higher plasma epinephrine levels (60 min: CON, 1.96 ± 0.29 vs LG, 4.25 ± 0.60 nM, P<0.05) compared with CON. Despite the attenuated HSL activity in LG, IMTG was decreased by exercise (0 min: 27.1 ± 2.0; 60 min: 22.5 ± 2.0 mmol.kg–1 dm, P<0.05), whereas no net reduction occurred in CON. To confirm the apparent effect of AMPK on HSL activity, we performed experiments in muscle cell culture. The epineprine‐induced increase in HSL activity was totally attenuated (P<0.05) by AICAR administration in L6 myotubes. These data provide new evidence indicating that AMPK is a major regulator of skeletal muscle HSL activity that can override β‐adrenergic stimulation. However, the increased IMTG degradation in LG suggests factors other than HSL activity are important for IMTG degradation.

Screening for the efficacy on lipid accumulation in 3T3-L1 cells is an effective tool for the identification of new anti-diabetic compounds

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, p < 0.001) and a fourfold improvement in TG extraction recovery rate (88-95%, p < 0.001) 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 screened >200 new candidates selected from Chinese medicine and identified 49 compounds from different classes which reduced TG content by >50% at 1 μM or >75% 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.

Neonatal overfeeding attenuates acute central pro-inflammatory effects of short-term high fat diet

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.

IRE1 impairs insulin signaling transduction of fructose-fed mice via JNK independent of excess lipid

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.

Fenofibrate insulates diacylglycerol in lipid droplet/ER and preserves insulin signaling transduction in the liver of high fat fed mice

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<0.01) into intermediate lipid metabolites, suggesting a FA oxidation-synthesis cycling in operation. Associated with these effects, diacylglycerols (DAGs) were sequestered within the lipid droplet/ER compartment, thus reducing their deposition in the cellular membrane, which is known to impair insulin signal transduction. These findings suggest that the reduction in membrane DAGs (rather than total hepatic steatosis) may be critical for the protection by fenofibrate-induced PPARα activation against hepatic insulin resistance induced by dietary fat.

System-L amino acid transporters play a key role in pancreatic β-cell signalling and function

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.

Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy

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. FASEB J. 30, 2549‐2556 (2016). www.fasebj.org

Angiotensin II causes b-cell dysfunction through an er stress-induced proinflammatory response

&NA; 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 &bgr;‐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 &bgr;‐cell dysfunction. Islets of Langerhans were isolated from C57BL/6J mice that had been infused with AngII in the presence or absence of taurine‐conjugated ursodeoxycholic acid (TUDCA) and effects on endoplasmic reticulum (ER) stress, inflammation, and &bgr;‐cell function determined. The mechanism of action of AngII was further investigated using isolated murine islets and clonal &bgr; cells. We show that AngII triggers ER stress, an increase in the messenger RNA expression of proinflammatory cytokines, and promotes &bgr;‐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 &bgr;‐cell dysfunction and indicates how therapies that promote ER homeostasis may be beneficial in the prevention of type 2 diabetes.