Attenuated Insulin Signaling Research Articles

Hepatic Dyrk1b impairs systemic glucose homeostasis by modulating Wbp2 expression in a kinase activity-dependent manner

Patients with gain-of-function mutations of Dyrk1b have higher fasting blood glucose (FBG) levels. However, the role of Dyrk1b in glucose metabolism is not fully elucidated. Herein, we found that hepatic Dyrk1b overexpression in mice impaired systemic glucose tolerance and hepatic insulin signaling. Dyrk1b overexpression in vitro attenuated insulin signaling in a kinase activity-dependent manner, and its kinase activity was required for its effect on systemic glucose homeostasis and hepatic insulin signaling in vivo. Dyrk1b ablation improved systemic glucose tolerance and hepatic insulin signaling in mice. Quantitative proteomic analyses showed that Dyrk1b downregulated WW domain-binding protein 2 (Wbp2) protein abundance. Mechanistically, Dyrk1b enhanced Wbp2 ubiquitylation and proteasomal degradation. Restoration of hepatic Wbp2 partially rescued the impaired glucose homeostasis in Dyrk1b overexpression mice. In addition, Dyrk1b inhibition with AZ191 moderately improved systemic glucose homeostasis. Our study uncovers that hepatic Dyrk1b impairs systemic glucose homeostasis via its modulation of Wbp2 expression in a kinase activity-dependent manner.

Tryptophanylation of insulin receptor by WARS attenuates insulin signaling.

Increased circulating amino acid levels have been linked to insulin resistance and development of type 2 diabetes (T2D), but the underlying mechanism remains largely unknown. Herein, we show that tryptophan modifies insulin receptor (IR) to attenuate insulin signaling and impair glucose uptake. Mice fed with tryptophan-rich chow developed insulin resistance. Excessive tryptophan promoted tryptophanyl-tRNA synthetase (WARS) to tryptophanylate lysine 1209 of IR (W-K1209), which induced insulin resistance by inhibiting the insulin-stimulated phosphorylation of IR, AKT, and AS160. SIRT1, but not other sirtuins, detryptophanylated IRW-K1209 to increase the insulin sensitivity. Collectively, we unveiled the mechanisms of how tryptophan impaired insulin signaling, and our data suggested that WARS might be a target to attenuate insulin resistance in T2D patients.

Metabolic consequences of skeletal muscle- and liver-specific BBSome deficiency.

The BBSome is a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins including BBS1. Humans and mice lacking a functional BBSome display obesity and type 2 diabetes, highlighting the importance of this protein complex for metabolic regulation. However, the contribution of the BBSome in insulin-sensitive tissues such as skeletal muscle and liver to metabolic regulation is ill-defined. Here, we show that disruption of the BBSome through Bbs1 gene deletion in the skeletal muscle had no effect on body weight or glucose handling, but improved insulin sensitivity of female mice without changing insulin receptor signaling. Interestingly, when fed an obesogenic diet, male mice lacking the Bbs1 gene in skeletal muscle exhibited heightened insulin sensitivity despite the comparable weight gain and glucose tolerance relative to controls. On the other hand, normal chow-fed mice missing the Bbs1 gene in hepatocytes displayed increased body weight, as well as impaired glucose handling and insulin sensitivity. This was associated with attenuated insulin signaling in liver and hepatocytes, but not skeletal muscle and white adipose tissue. Moreover, hepatocytes lacking the Bbs1 gene displayed significant reduction in plasma membrane insulin receptor levels due to the mitochondrial dysfunction evoked by loss of the BBSome. Together, these findings demonstrate that myocyte BBSome is minimally involved in metabolic regulation, whereas the hepatic BBSome plays a critical role in the control of energy homeostasis and insulin sensitivity through its requirement for insulin receptor trafficking.NEW & NOTEWORTHY The ongoing epidemic of obesity and associated illnesses highlights the need to understand the biological processes that regulate energy balance. Here, we identified an important role for a protein complex called BBSome in the control of hepatic function. We show that the liver BBSome is necessary to maintain body weight and blood glucose levels due to its requirements to generate energy and detect insulin, a hormone that is essential for metabolic regulation.

MicroRNA-34a Mediates High-Fat-Induced Hepatic Insulin Resistance by Targeting ENO3

The etiology of numerous metabolic disorders is characterized by hepatic insulin resistance (IR). Uncertainty surrounds miR-34a’s contribution to high-fat-induced hepatic IR and its probable mechanism. The role and mechanism of miR-34a and its target gene ENO3 in high-fat-induced hepatic IR were explored by overexpressing/suppressing miR-34a and ENO3 levels in in vivo and in vitro experiments. Moreover, as a human hepatic IR model, the miR-34a/ENO3 pathway was validated in patients with non-alcoholic fatty liver disease (NAFLD). The overexpression of hepatic miR-34a lowered insulin signaling and altered glucose metabolism in hepatocytes. In contrast, reducing miR-34a expression significantly reversed hepatic IR indices induced by palmitic acid (PA)/HFD. ENO3 was identified as a direct target gene of miR-34a. Overexpression of ENO3 effectively inhibited high-fat-induced hepatic IR-related indices both in vitro and in vivo. Moreover, the expression patterns of members of the miR-34a/ENO3 pathway in the liver tissues of NAFLD patients was in line with the findings of both cellular and animal studies. A high-fat-induced increase in hepatic miR-34a levels attenuates insulin signaling and impairs glucose metabolism by suppressing the expression of its target gene ENO3, ultimately leading to hepatic IR. The miR-34a/ENO3 pathway may be a potential therapeutic target for hepatic IR and related metabolic diseases.

Ganglioside GM3 prevents high fat diet-induced hepatosteatosis via attenuated insulin signaling pathway.

Gangliosides, sialic acid-containing glycosphingolipids, are widely involved in regulations of signal transductions to control cellular functions. It has been suggested that GM3, the simplest structure among gangliosides, is involved in insulin resistance, whereas it remains unclear whether insulin signaling diminished by GM3 actually aggravates the pathological conditions in metabolic disorders. Moreover, the functional roles of gangliosides in the regulation of insulin signaling have not yet been fully elucidated in liver or hepatocytes despite that it is one of the major insulin-sensitive organs. To understand physiological roles of GM3 in metabolic homeostasis in liver, we conducted a high fat diet (HFD) loading experiment using double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase, which lack all gangliosides except GM3, as well as wild-type (WT) mice. DKO mice were strikingly resistant to HFD-induced hepatosteatosis, and hepatic lipogenesis-related molecules including insulin signaling components were down-regulated in HFD-fed DKO. Furthermore, we established primary hepatocyte cultures from DKO and WT mice, and examined their responses to insulin in vitro. Following insulin stimulation, DKO hepatocytes expressing GM3 showed attenuated expression and/or activations in the downstream components compared with WT hepatocytes expressing GM2. While insulin stimulation induced lipogenic proteins in hepatocytes from both genotypes, their expression levels were lower in DKO than in WT hepatocytes after insulin treatment. All our findings suggest that the modified gangliosides, i.e., a shift to GM3 from GM2, might exert a suppressive effect on lipogenesis by attenuating insulin signaling at least in mouse hepatocytes, which might result in protection of HFD-induced hepatosteatosis.

FoxO-KLF15 pathway switches the flow of macronutrients under the control of insulin

SummaryKLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1/3 transcriptionally regulates Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis combined with the genome-wide transcription-factor-screening method “TFEL scan”, using our original Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased via FoxOs by attenuating insulin signaling. Furthermore, FoxOs elevate the expression levels of amino acid catabolic enzymes and suppress SREBP-1c via KLF15, resulting in accelerated amino acid breakdown and suppressed lipogenesis during fasting. Thus, the FoxO-KLF15 pathway contributes to switching the macronutrient flow in the liver under the control of insulin.

Metabolic Differences between Subcutaneous and Visceral Adipocytes Differentiated with an Excess of Saturated and Monounsaturated Fatty Acids.

Obesity is a major health problem in highly industrialized countries. High-fat diet (HFD) is one of the most common causes of obesity and obesity-related disorders. There are considerable differences between fat depots and the corresponding risks of metabolic disorders. We investigated the various effects of an excess of fatty acids (palmitic 16:0, stearic 18:0, and oleic acids 18:1n−9) on adipogenesis of subcutaneous- and visceral-derived mesenchymal stem cells (MSCs) and phenotypes of mature adipocytes. MSCs of white adipose tissue were acquired from adipose tissue biopsies obtained from subcutaneous and visceral fat depots from patients undergoing abdominal surgery. The MSCs were extracted and differentiated in vitro with the addition of fatty acids. Oleic acid stimulated adipogenesis, resulting in higher lipid content and larger adipocytes. Furthermore, oleic acid stimulated adipogenesis by increasing the expression of CCAAT enhancer binding protein β (CEBPB) and peroxisome proliferator activated receptor γ (PPARG). All of the examined fatty acids attenuated the insulin-signaling pathway and radically reduced glucose uptake following insulin stimulation. Visceral adipose tissue was shown to be more prone to generate inflammatory stages. The subcutaneous adipose tissue secreted a greater quantity of adipokines. To summarize, oleic acid showed the strongest effect on adipogenesis. Furthermore, all of the examined fatty acids attenuated insulin signaling and secretion of cytokines and adipokines.

The Kinase Activity of Drosophila BubR1 Is Required for Insulin Signaling-Dependent Stem Cell Maintenance

As a core component of the mitotic checkpoint complex, BubR1 has a modular organization of molecular functions, with KEN box and other motifs at the N terminus inhibiting the anaphase-promoting complex/cyclosome, and a kinase domain at the C terminus, whose function remains unsettled, especially at organismal levels. We generate knock-in BubR1 mutations in the Drosophila genome to separately disrupt the KEN box and the kinase domain. All of the mutants are homozygously viable and fertile and show no defects in mitotic progression. The mutants without kinase activity have an increased lifespan and phenotypic changes associated with attenuated insulin signaling, including reduced InR on the cell membrane, weakened PI3K and AKT activity, and elevated expression of dFoxO targets. The BubR1 kinase-dead mutants have a reduced cap cell number in female germaria, which can be rescued by expressing a constitutively active InR. We conclude that one major physiological role of BubR1 kinase in Drosophila is to modulate insulin signaling.

2010-P: Adipocyte Clusterin Is a Link between Cardiometabolism and Alzheimer’s Disease

Background: Obesity heightens the risk for both cardiometabolic complications and Alzheimer’s disease (AD). Although the reasons have yet to be fully elucidated, weight loss improves cognition. Given this link, factors overproduced by obese adipose tissue are ideal candidates for novel investigation into AD pathogenesis. We have recently shown that clusterin from the adipocyte is related to multiple factors of the metabolic syndrome (insulin resistance, dyslipidemia, NAFLD, and atherosclerosis). Clusterin is also involved in CNS lipid trafficking and has been proposed as an AD biomarker. Rationale: Our goals were to characterize the effect of high-fat diet (HFD) feeding on CNS clusterin and neuronal insulin signaling in an AD mouse model (5XFAD); and furthermore, determine the relationship of adipocyte clusterin with neurocognition and insulin sensitivity in obese humans at baseline and following bariatric surgery. Methods: We treated 5XFAD and WT mice with normal chow and HFD for 4 mo., measured CNS clusterin, and determined its effect on neuronal insulin signaling. We next measured subcutaneous adipocyte (SAd) clusterin expression, and performed neuropsychological evaluation and a hyperinsulinemic-euglycemic clamp procedure in obese human subjects (n=11) before and after sleeve gastrectomy. Results: In the 5XFAD mice, HFD increases brain clusterin levels compared to WT, and in primary cortical neurons clusterin attenuates insulin signaling in a dose-dependent manner. In humans, SAd clusterin is related to hepatic insulin resistance, higher fasting glucose, and reduced working memory/attention. After sleeve gastrectomy SAd clusterin expression is reduced, with a significant inverse association between clusterin and working memory/attention and hepatic insulin sensitivity. Conclusions: Our data suggests that SAd clusterin is a potential player in AD pathology, providing a critical link between excess adiposity, T2D, insulin resistance, and heightened AD risk. Disclosure D. Bradley: None. J.Z. Liu: None. A.M. Blaszczak: None. V.P. Wright: None. R. Tarawneh: None. Y. Liu: None. J. Ryu: None. S. Yoon: None. W. Hsueh: None. Funding American Diabetes Association (1-16-ICTS-049 to W.H.); National Institutes of Health (KL2TR001068, R01HL135622)

Insulin deficiency promotes formation of toxic amyloid-β42 conformer co-aggregating with hyper-phosphorylated tau oligomer in an Alzheimer's disease model

The toxic conformer of amyloid β-protein (Aβ) ending at 42 (Aβ42), which contains a unique turn conformation at amino acid residue positions 22 and 23 and tends to form oligomers that are neurotoxic, was reported to play a critical role in the pathomechanisms of Alzheimer's disease (AD), in which diabetes mellitus (DM)-like mechanisms are also suggested to be operative. It remains to be established whether the attenuation of insulin signaling is involved in an increase of toxic Aβ42 conformer levels. The present study investigated the association between impaired insulin metabolism and formation of toxic Aβ42 conformers in the brains of an AD mouse model. In particular, we studied whether insulin deficiency or resistance affected the formation of toxic Aβ42 conformers in vivo. We induced insulin deficiency and resistance in 3xTg-AD mice, a mouse AD model harboring two familial AD-mutant APP (KM670/671NL) and PS1 (M146 V) genes and a mutant TAU (P301L) gene, by streptozotocin (STZ) injection and a high fructose diet (HFuD), respectively. Cognitive impairment was significantly worsened by STZ injection but not by HFuD. Dot blot analysis revealed significant increases in total Aβ42 levels and the ratio of toxic Aβ42 conformer/total Aβ42 in STZ-treated mice compared with control and HFuD-fed mice. Immunostaining showed the accumulation of toxic Aβ42 conformers and hyper-phosphorylated tau protein (p-tau), which was more prominent in the cortical and hippocampal neurons of STZ-treated mice compared with HFuD-fed and control mice. HFuD-fed mice showed only a mild-to-moderate increase of these proteins compared with controls. Toxic Aβ42 conformers were co-localized with p-tau oligomers (Pearson's correlation coefficient = 0.62) in the hippocampus, indicating their co-aggregation. Toxic Aβ42 conformer levels were inversely correlated with pancreatic insulin secretion capacity as shown by fasting immunoreactive insulin levels in STZ-treated mice (correlation coefficient = −0.5879, p = .04441), but not HFuD-fed mice, suggesting a decrease in serum insulin levels correlates with toxic Aβ42 conformer formation. Levels of p-Akt and phosphorylated glycogen synthase kinase-3β measured by a homogeneous time-resolved fluorescence assay were significantly lower in STZ-treated mice than in HFuD-fed mice, suggesting a greater inhibition of brain insulin signaling by STZ than HFuD, although both levels were significantly decreased in these groups compared with controls. Iba1-positive and NOS2-positive areas in the cortex and hippocampus were significantly increased in STZ-treated mice and to a lesser extent in HFuD-fed mice compared with controls. These findings suggest that insulin deficiency rather than insulin resistance and the resultant impairment of brain insulin signaling facilitates the formation of toxic Aβ42 conformer and its co-aggregation with p-tau oligomers, and that insulin deficiency is an important pathogenic factor in the progression of AD.

Aerobic Exercise Improves Food Reward Systems in Obese Rats via Insulin Signaling Regulation of Dopamine Levels in the Nucleus Accumbens.

The dopaminergic pathway, comprising projections from the ventral tegmental area to the nucleus accumbens, constitutes the core of the brain reward system. Insufficient food reward caused by dopamine signaling dysfunction in the nucleus accumbens is an important contributor to obesity and may be associated with insulin signaling. Aerobic exercise has a positive effect on both preventing and treating obesity. In addition, physical exercise is important in striatal dopamine homeostasis and improves insulin sensitivity in the peripheral and central nervous system. Therefore, we hypothesized that aerobic exercise may increase dopamine levels in the nucleus accumbens through insulin signaling, thus improving food reward in obesity. In the present study, we used a rat model of obesity, induced by high fat diet. Obese rats exhibited lower basic dopamine concentration in the nucleus accumbens induced by eating or extracellular insulin, attenuated insulin signaling, and increased fat preference. Interestingly, an 8-week aerobic exercise regimen reversed these symptoms. In addition, we noted a significant increase in insulin Akt/GSK3-β signal transduction in the nucleus accumbens. These data demonstrate that aerobic exercise promotes dopamine levels in the nucleus accumbens through insulin signal transduction, which may constitute an important neurobiological mechanism of exercise against obesity.

Asprosin attenuates insulin signaling pathway through PKCδ-activated ER stress and inflammation in skeletal muscle.

It has been reported that asprosin is a novel adipokine which is augmented in mice and humans with type 2 diabetes (T2DM). Asprosin stimulates hepatic gluconeogenesis under fasting conditions. However, the roles of asprosin in inflammation, endoplasmic reticulum (ER) stress, and insulin resistance in skeletal muscle has not been studied. In the currents study, elevated levels of asprosin expression were observed in adipocytes under hyperlipidemic conditions. Treatment of C2C12 myocytes with asprosin-induced ER stress markers (phosphorylated inositol-requiring enzyme 1 and eukaryotic initiation factor 2, and CHOP expression) as well as inflammation markers (interleukin-6 expression, phosphorylated IκB, and nuclear translocated nuclear factor-κβ). Finally, asprosin treatment promoted exacerbation of insulin sensitivity as determined by levels of insulin receptor substrate 1 and Akt phosphorylation as well as glucose uptake. Moreover, treatment of asprosin augmented protein kinase C-δ (PKCδ) phosphorylation and nuclear translocation, but suppressed messenger RNA expression of sarcoplasmic reticulum Ca2+ ATPase 2b in both C2C12 myocytes and in mouse soleus skeletal muscle. These asprosin-induced effects were markedly decreased in small interfering (si) RNA-mediated PKCδ-knockdown in C2C12 myocytes. These results suggest that asprosin results in impairment of insulin sensitivity in skeletal muscle through PKCδ-associated ER stress/inflammation pathways and may be a valuable strategy for management of insulin resistance and T2DM.

OR05-4 Androgen Induced Metabolic Dysfunction in Female Mice is Prevented by Deletion of Liver Androgen Receptor

Women with elevated androgen often exhibit hyperinsulinemia and insulin resistance. Many models have been created to produce metabolic dysfunction by manipulating androgen levels. Previously we reported that hyperandrogenemia produced by implantation of a DHT pellet may induce whole body insulin resistance through hepatic androgen receptor (AR) in vitro. Therefore, we conditionally disrupted the hepatic AR both developmentally (by breeding the albumin-CRE mouse (alb-CRE; liver-specific Cre expression) with ARfl/fl mice (LivARKO)) and acutely (by tail vein injection in ARfl/fl mice of adeno-associated virus with albumin promoter driven Cre vector (adLivARKO)). Impaired glucose tolerance, pyruvate tolerance, and insulin tolerance were prevented in both LivARKO-DHT and adLivARKO-DHT mice compared to control mice treated with DHT (ARfl/fl; cre-; Con-DHT) mice. Further, the glucose infusion rate is significantly reduced in LivARKO-DHT mice compared to Con-DHT mice as measured during hyperinsulinemia-euglycemia clamp suggesting improved insulin-induced glucose disposal. We observed that primary hepatocytes treated with DHT from both women and female mice showed reduced PI3K-pAKT and pFOXO1 activation by insulin, and increased ex vivo glucose production. LivARKO primary hepatocytes were resistant to DHT induced attenuated insulin signaling and increased gluconeogenesis (ex vivo) and this was confirmed in LivARKO in vivo. These data support a paradigm in which in females, elevated androgen disrupts metabolic function via hepatic AR.

Overexpression of Long-Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Oxidation and Free Radical Formation While Attenuating Insulin Signaling in Primary Human Skeletal Myotubes.

In rodent skeletal muscle, acyl-coenzyme A (CoA) synthetase 5 (ACSL-5) is suggested to localize to the mitochondria but its precise function in human skeletal muscle is unknown. The purpose of these studies was to define the role of ACSL-5 in mitochondrial fatty acid metabolism and the potential effects on insulin action in human skeletal muscle cells (HSKMC). Primary myoblasts isolated from vastus lateralis (obese women (body mass index (BMI) = 34.7 ± 3.1 kg/m2)) were transfected with ACSL-5 plasmid DNA or green fluorescent protein (GFP) vector (control), differentiated into myotubes, and harvested (7 days). HSKMC were assayed for complete and incomplete fatty acid oxidation ([1-14C] palmitate) or permeabilized to determine mitochondrial respiratory capacity (basal (non-ADP stimulated state 4), maximal uncoupled (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-linked) respiration, and free radical (superoxide) emitting potential). Protein levels of ACSL-5 were 2-fold higher in ACSL-5 overexpressed HSKMC. Both complete and incomplete fatty acid oxidation increased by 2-fold (p < 0.05). In permeabilized HSKMC, ACSL-5 overexpression significantly increased basal and maximal uncoupled respiration (p < 0.05). Unexpectedly, however, elevated ACSL-5 expression increased mitochondrial superoxide production (+30%), which was associated with a significant reduction (p < 0.05) in insulin-stimulated p-Akt and p-AS160 protein levels. We concluded that ACSL-5 in human skeletal muscle functions to increase mitochondrial fatty acid oxidation, but contrary to conventional wisdom, is associated with increased free radical production and reduced insulin signaling.

Clusterin Impairs Hepatic Insulin Sensitivity and Adipocyte Clusterin Associates With Cardiometabolic Risk.

Components of the adipose tissue (AT) extracellular matrix (ECM) are recently discovered contributors to obesity-related cardiometabolic disease. We identified increased adipocyte expression of ECM-related clusterin (apolipoprotein J) in obese versus lean women by microarray. Our objective was to determine 1) whether subcutaneous AT adipocyte (SAd) clusterin and serum clusterin are associated with insulin resistance (IR) and known markers of cardiometabolic risk and 2) how clusterin may contribute to increased risk. We validated increased clusterin expression in adipocytes from a separate group of 18 lean and 54 obese individuals. The relationship of clusterin gene expression and plasma clusterin with IR, cardiovascular biomarkers, and risk of cardiovascular disease (CVD) was then determined. Further investigations in human cultured cells and in aged LDLR-/- mice prone to development of obesity-associated complications were performed. SAd clusterin correlated with IR, multiple CVD biomarkers, and CVD risk, independent of traditional risk factors. Circulating human clusterin exhibited similar associations. In human adipocytes, palmitate enhanced clusterin secretion, and in human hepatocytes, clusterin attenuated insulin signaling and APOA1 expression and stimulated hepatic gluconeogenesis. LRP2 (megalin), a clusterin receptor, highly expressed in liver, mediated these effects, which were inhibited by LRP2 siRNA. In response to Western diet feeding, an increase in adipocyte clusterin expression was associated with a progressive increase in liver fat, steatohepatitis, and fibrosis in aged LDLR-/- mice. Adipocyte-derived clusterin is a novel ECM-related protein linking cardiometabolic disease and obesity through its actions in the liver.

Tumour necrosis factor α induces neuroinflammation and insulin resistance in immortalised hypothalamic neurones through independent pathways.

The links between obesity, inflammation and insulin resistance, which are all key characteristics of type 2 diabetes mellitus, are yet to be delineated in the brain. One of the key neuroinflammatory proteins detected in the hypothalamus with over-nutrition is tumour necrosis factor (TNF)α. Using immortalised embryonic rat and mouse hypothalamic cell lines (rHypoE-7 and mHypoE-46) that express orexigenic neuropeptide Y and agouti-related peptide, we investigated changes in insulin signalling and inflammatory gene marker mRNA expression after TNFα exposure. A quantitative polymerase chain reaction array of 84 inflammatory markers (cytokines, chemokines and receptors) demonstrated an increase in the expression of multiple genes encoding inflammatory markers upon exposure to 100ngmL-1 TNFα for 4hours. Furthermore, neurones pre-exposed to TNFα (50ngmL-1 ) for 6 or 16hours exhibited a significant reduction in phosphorylated Akt compared to control after insulin treatment, indicating the attenuation of insulin signalling. mRNA expression of insulin signalling-related genes was also decreased with exposure to TNFα. TNFα significantly increased mRNA expression of IκBα, Tnfrsf1a and IL6 at 4 and 24hours, activating a pro-inflammatory state. An inhibitor study using an inhibitor of nuclear factor kappa B kinase subunit β (IKK-β) inhibitor, PS1145, demonstrated that TNFα-induced neuroinflammatory marker expression occurs through the IKK-β/nuclear factor-kappa B pathway, whereas oleate, a monounsaturated fatty acid, had no effect on inflammatory markers. To test the efficacy of anti-inflammatory treatment to reverse insulin resistance, neurones were treated with TNFα and PS1145, which did not significantly restore the TNFα-induced changes in cellular insulin sensitivity, indicating that an alternative pathway may be involved. In conclusion, exposure to the inflammatory cytokine TNFα causes cellular insulin resistance and inflammation marker expression in the rHypoE-7 and mHypoE-46 neurones, consistent with effects seen with TNFα in peripheral tissues. It also mimics insulin- and palmitate-induced insulin resistance in hypothalamic neurones. The present study provides further evidence that altered central energy metabolism may be caused by obesity-induced cytokine expression.

169 - Oxidized neutral lipid lipolysis regulates insulin signaling during acute stress

Acute hyperglycemia and systemic insulin resistance (IR) often develop after injury or surgery. This stress response, appropriately known as critical illness diabetes, leads to increased post-operative complications and mortality. Insulin administration reduces this hyperglycemia in some patients but has a risk of dangerous hypoglycemia. The underlying mechanisms driving IR are unknown, which limits the development of alternative therapies and emphasizes the need to elucidate the pathophysiology. It is, however, known that surgical animal models rapidly develop adipose IR, and impaired insulin action in adipose alone can result in whole-body IR. Therefore, we focused on how adipose contributes to stress-induced IR. We have previously demonstrated using a mouse model of surgery that the development of IR and hyperglycemia are dependent on adipose lipolysis. Furthermore, β-adrenergic stimulation of adipocytes causes inhibition and dissociation of mTORC1 and mTORC2, key steps in the insulin cascade, in a lipolysis-dependent manner. Therefore, we hypothesized that a product of adipose lipolysis was responsible for the development of IR. We found that lipid extracts isolated from forskolin-stimulated 3T3-L1 adipocytes inhibited the activity of mTORC in vitro, however “traditional” lipolytic products (DAG, MAG, fatty acids) had no effect. Interestingly, stimulating peroxidation of fatty acids further exacerbated mTORC inhibition, while antioxidants reversed this effect. This suggests that the active species is an oxidized fatty acid. In fact, incubation of mTORC with in vitro auto-oxidized fatty acids is sufficient to inhibit kinase activity. Therefore, we propose that lipolysis breaks down oxidized TAGs into oxidized fatty acids, which cause mTORC dissociation and attenuation of insulin signaling during the stress response. Our work will elucidate a novel role for oxidized TAGs and their lipolytic products in the regulation of insulin signaling. Defining the mechanism by which catecholamine-stimulated lipolysis attenuates insulin signaling will provide novel therapeutic targets for improved post-operative glucose homeostasis.

Impact of Fetuin-A (AHSG) on Tumor Progression and Type 2 Diabetes.

Fetuin-A is the protein product of the AHSG gene in humans. It is mainly synthesized by the liver in adult humans and is secreted into the blood where its concentration can vary from a low of ~0.2 mg/mL to a high of ~0.8 mg/mL. Presently, it is considered to be a multifunctional protein that plays important roles in diabetes, kidney disease, and cancer, as well as in inhibition of ectopic calcification. In this review we have focused on work that has been done regarding its potential role(s) in tumor progression and sequelae of diabetes. Recently a number of laboratories have demonstrated that a subset of tumor cells such as pancreatic, prostate and glioblastoma multiform synthesize ectopic fetuin-A, which drives their progression. Fetuin-A that is synthesized, modified, and secreted by tumor cells may be more relevant in understanding the pathophysiological role of this enigmatic protein in tumors, as opposed to the relatively high serum concentrations of the liver derived protein. Lastly, auto-antibodies to fetuin-A frequently appear in the sera of tumor patients that could be useful as biomarkers for early diagnosis. In diabetes, solid experimental evidence shows that fetuin-A binds the β-subunit of the insulin receptor to attenuate insulin signaling, thereby contributing to insulin resistance in type 2 diabetes mellitus (T2DM). Fetuin-A also may, together with free fatty acids, induce apoptotic signals in the beta islets cells of the pancreas, reducing the secretion of insulin and further exacerbating T2DM.

Abstract 377: Time-restricted feeding: A dietary intervention to treat breast cancer in postmenopausal obese mice

Abstract Background: Abundant evidence indicates that obesity increases risk for breast cancer and the incidence further increases by 40% in obese postmenopausal women. Therefore it is envisioned by diverse research groups that the risk of breast cancer in obese postmenopausal women is an ever-increasing menace that needs to be curbed soon with effective practical strategies that could have a far-reaching impact. In this setting, time-restricted feeding (TRF), the practice of consuming ad libitum energy during the normal active phase, has been demonstrated to reinforce normal metabolic regulation, thereby attenuating obesity-driven metabolic deregulation. Although studies have assessed the effect of TRF on metabolism, no investigations have been carried out in human or mouse exploring TRF in cancer remission. Therefore, the present work is to understand the efficacy of TRF for improved breast cancer remission in postmenopausal obese female mice. Further, the work explores the mechanistic link of TRF for reduced tumor growth. Methods: Ovariectomized and 4-vinylcyclohexene diepoxide (VCD) treated mice were used as a postmenopausal model. Both ovariectomized and VCD mice were made obese by feeding them with 60% high-fat diet (HFD) and then grouped into ad libitum group (24 h access to food) and TRF group (8 hr access to food at night) to assess metabolic and tumor growth effect of TRF. To develop breast tumor, mice were injected with py230 breast cancer cell line into the mammary fat pad. The metabolic effect of TRF was assessed by glucose tolerance test, insulin tolerance test, body weight measurement, food intake, lipid accumulation in liver by HE staining and measurement of different tissue weight. Measuring the tumor volume over time and tumor weight assessed TRF effect on tumor growth. Performing a tumor growth study in mice fed with HFD and HFD containing diazoxide assessed insulin dependency of tumor growth in ad libitum group. Insulin-dependent tumor growth was validated by tumor growth study in normal chow-fed mice implanted with insulin pump or without pump (control). Results: The preliminary studies in ovariectomized and VCD-treated postmenopausal mice suggest that restricting access to Western-style HFD in active night phase improves insulin resistance, glucose tolerance, and hepatic steatosis. Further, TRF exhibited reduced tumor growth compared to ad libitum group. More importantly, the results from tumor growth study in mice fed with HFD with/without diazoxide or normal chow mice with/without insulin pump, suggest that the tumor growth is insulin dependent and TRF may be acting through attenuating insulin signaling. Conclusion: Experimental and animal model data corroborate that TRF improves metabolic deregulation and reduces breast tumor growth in HFD-fed obese mice. The results suggest putative application of such therapeutic intervention for breast cancer therapy. Citation Format: Manasi Das, Emilie Gross, Deepak Kumar, Consuelo Sauceda, Hyun-Tae Park, Dorothy D. Sears, Lesley Ellies, Nicholas Webster. Time-restricted feeding: A dietary intervention to treat breast cancer in postmenopausal obese mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 377.

A High‐Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase

Phosphoenolpyruvate Carboxykinase (PEPCK EC 4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the presence of GTP. In humans, two isoforms of PEPCK are found: cytosolic form (PEPCK‐C, also called PCK1) and mitochondrial form (PEPCK‐M, also called PCK2). PEPCK‐C (PCK1) is a rate‐controlling enzyme in gluconeogenesis. Recent studies found abnormal concentrations of PEPCK in diabetic mice. Overexpression of PEPCK attenuates insulin signaling and decreases hepatic insulin sensitivity in transgenic mice. Our high throughput assay for Phosphoenolpyruvate Carboxykinase activity provides a valuable tool for both mechanistic and therapeutic studies of diabetes as well as avenue for targeting PEPCK activity.The Phosphoenolpyruvate Carboxykinase activity assay is developed by using a set of specific enzymes that convert Phosphoenolpyruvate (PEP) and carbonate into a series of intermediates and hydrogen peroxide, which in turn, reacts with a probe generating a colorimetric signal (OD570 nm). The color intensity is directly proportional to the amount of active Phosphoenolpyruvate Carboxykinase. This assay is simple, sensitive, high‐throughput adaptable and can detect less than 10 μU of Phosphoenolpyruvate Carboxykinase activity per sample.In conclusion, a Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay kit is developed. The kit provides easy and robust tool for measuring PEPCK activity in biological and purified samples (BioVision, Inc. Cat. # K359‐100).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.