Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Chronic hyperglycaemia increases the vulnerability of the hippocampus to oxidative damage induced during post-hypoglycaemic hyperglycaemia in a mouse model of chemically induced type 1 diabetes.

AIMS/HYPOTHESIS: Chronic hyperglycaemia and recurrent hypoglycaemia are independently associated with accelerated cognitive decline in type 1 diabetes. Recurrent hypoglycaemia in rodent models of chemically induced (streptozotocin [STZ]) diabetes leads to cognitive impairment in memory-related tasks associated with hippocampal oxidative damage. This study examined the hypothesis that post-hypoglycaemic hyperglycaemia in STZ-diabetes exacerbates hippocampal oxidative stress and explored potential contributory mechanisms. METHODS: The hyperinsulinaemic glucose clamp technique was used to induce equivalent hypoglycaemia and to control post-hypoglycaemic glucose levels in mice with and without STZ-diabetes and Nrf2-/- mice (lacking Nrf2 [also known as Nfe2l2]). Subsequently, quantitative proteomics based on stable isotope labelling by amino acids in cell culture and biochemical approaches were used to assess oxidative damage and explore contributory pathways. RESULTS: Evidence of hippocampal oxidative damage was most marked in mice with STZ-diabetes exposed to post-hypoglycaemic hyperglycaemia; these mice also showed induction of Nrf2 and the Nrf2 transcriptional targets Sod2 and Hmox-1. In this group, hypoglycaemia induced a significant upregulation of proteins involved in alternative fuel provision, reductive biosynthesis and degradation of damaged proteins, and a significant downregulation of proteins mediating the stress response. Key differences emerged between mice with and without STZ-diabetes following recovery from hypoglycaemia in proteins mediating the stress response and reductive biosynthesis. CONCLUSIONS/INTERPRETATION: There is a disruption of the cellular response to a hypoglycaemic challenge in mice with STZ-induced diabetes that is not seen in wild-type non-diabetic animals. The chronic hyperglycaemia of diabetes and post-hypoglycaemic hyperglycaemia act synergistically to induce oxidative stress and damage in the hippocampus, possibly leading to irreversible damage/modification to proteins or synapses between cells. In conclusion, recurrent hypoglycaemia in sub-optimally controlled diabetes may contribute, at least in part, to accelerated cognitive decline through amplifying oxidative damage in key brain regions, such as the hippocampus. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in ProteomeXchange, accession no. 1-20220824-173727 ( www.proteomexchange.org ). Additional datasets generated during and/or analysed during the present study are available from the corresponding author upon reasonable request.

CD44 contributes to hyaluronan-mediated insulin resistance in skeletal muscle of high-fat-fed C57BL/6 mice.

Extracellular matrix hyaluronan is increased in skeletal muscle of high-fat-fed insulin-resistant mice, and reduction of hyaluronan by PEGPH20 hyaluronidase ameliorates diet-induced insulin resistance (IR). CD44, the main hyaluronan receptor, is positively correlated with type 2 diabetes. This study determines the role of CD44 in skeletal muscle IR. Global CD44-deficient (cd44-/-) mice and wild-type littermates (cd44+/+) were fed a chow diet or 60% high-fat diet for 16 wk. High-fat-fed cd44-/- mice were also treated with PEGPH20 to evaluate its CD44-dependent action. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (ICv). High-fat feeding increased muscle CD44 protein expression. In the absence of differences in body weight and composition, despite lower clamp insulin during ICv, the cd44-/- mice had sustained glucose infusion rate (GIR) regardless of diet. High-fat diet-induced muscle IR as evidenced by decreased muscle glucose uptake (Rg) was exhibited in cd44+/+ mice but absent in cd44-/- mice. Moreover, gastrocnemius Rg remained unchanged between genotypes on chow diet but was increased in high-fat-fed cd44-/- compared with cd44+/+ when normalized to clamp insulin concentrations. Ameliorated muscle IR in high-fat-fed cd44-/- mice was associated with increased vascularization. In contrast to previously observed increases in wild-type mice, PEGPH20 treatment in high-fat-fed cd44-/- mice did not change GIR or muscle Rg during ICv, suggesting a CD44-dependent action. In conclusion, genetic CD44 deletion improves muscle IR, and the beneficial effects of PEGPH20 are CD44-dependent. These results suggest a critical role of CD44 in promoting hyaluronan-mediated muscle IR, therefore representing a potential therapeutic target for diabetes.

AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner.

AIM: To test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. MATERIALS AND METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. RESULTS: In U373 cells, A-769662 (100 µM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 µM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.

Identification of caveolar resident proteins in ventricular myocytes using a quantitative proteomic approach: dynamic changes in caveolar composition following adrenoceptor activation.

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation-contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation. Rat ventricular myocytes were treated with methyl-ß-cyclodextrin (MßCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MßCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MßCD treatment. Selective activation of α-, ß1-, and ß2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes. We typically identified 600-850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.

Substrate recognition by the cell surface palmitoyl transferase DHHC5.

The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.

Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.

The electrogenic Na/Ca exchanger (NCX) mediates bidirectional Ca movements that are highly sensitive to changes of Na gradients in many cells. NCX1 is implicated in the pathogenesis of heart failure and a number of cardiac arrhythmias. We measured NCX1 palmitoylation using resin-assisted capture, the subcellular location of yellow fluorescent protein-NCX1 fusion proteins, and NCX1 currents using whole-cell voltage clamping. Rat NCX1 is substantially palmitoylated in all tissues examined. Cysteine 739 in the NCX1 large intracellular loop is necessary and sufficient for NCX1 palmitoylation. Palmitoylation of NCX1 occurs in the Golgi and anchors the NCX1 large regulatory intracellular loop to membranes. Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1. However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients. The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs. Thus, NCX palmitoylation ubiquitously modulates Ca homeostasis and membrane domain function in cells that express NCX proteins.

AMPK: regulating energy balance at the cellular and whole body levels.

AMP-activated protein kinase appears to have evolved in single-celled eukaryotes as an adenine nucleotide sensor that maintains energy homeostasis at the cellular level. However, during evolution of more complex multicellular organisms, the system has adapted to interact with hormones so that it also plays a key role in balancing energy intake and expenditure at the whole body level.

Altered amyloid precursor protein processing regulates glucose uptake and oxidation in cultured rodent myotubes.

AIMS/HYPOTHESIS: Impaired glucose uptake in skeletal muscle is an important contributor to glucose intolerance in type 2 diabetes. The aspartate protease, beta-site APP-cleaving enzyme 1 (BACE1), a critical regulator of amyloid precursor protein (APP) processing, modulates in vivo glucose disposal and insulin sensitivity in mice. Insulin-independent pathways to stimulate glucose uptake and GLUT4 translocation may offer alternative therapeutic avenues for the treatment of diabetes. We therefore addressed whether BACE1 activity, via APP processing, in skeletal muscle modifies glucose uptake and oxidation independently of insulin. METHODS: Skeletal muscle cell lines were used to investigate the effects of BACE1 and α-secretase inhibition and BACE1 and APP overexpression on glucose uptake, GLUT4 cell surface translocation, glucose oxidation and cellular respiration. RESULTS: In the absence of insulin, reduction of BACE1 activity increased glucose uptake and oxidation, GLUT4myc cell surface translocation, and basal rate of oxygen consumption. In contrast, overexpressing BACE1 in C2C12 myotubes decreased glucose uptake, glucose oxidation and oxygen consumption rate. APP overexpression increased and α-secretase inhibition decreased glucose uptake in C2C12 myotubes. The increase in glucose uptake elicited by BACE1 inhibition is dependent on phosphoinositide 3-kinase (PI3K) and mimicked by soluble APPα (sAPPα). CONCLUSIONS/INTERPRETATION: Inhibition of muscle BACE1 activity increases insulin-independent, PI3K-dependent glucose uptake and cell surface translocation of GLUT4. As APP overexpression raises basal glucose uptake, and direct application of sAPPα increases PI3K-protein kinase B signalling and glucose uptake in myotubes, we suggest that α-secretase-dependent shedding of sAPPα regulates insulin-independent glucose uptake in skeletal muscle.

Continuous hypothalamic K(ATP) activation blunts glucose counter-regulation in vivo in rats and suppresses K(ATP) conductance in vitro.

AIMS/HYPOTHESIS: Acute systemic delivery of the sulfonylurea receptor (SUR)-1-specific ATP-sensitive K(+) channel (K(ATP)) opener, NN414, has been reported to amplify glucose counter-regulatory responses (CRRs) in rats exposed to hypoglycaemia. Thus, we determined whether continuous NN414 could prevent hypoglycaemia-induced defective counter-regulation. METHODS: Chronically catheterised male Sprague-Dawley rats received a continuous infusion of NN414 into the third ventricle for 8 days after implantation of osmotic minipumps. Counter-regulation was examined by hyperinsulinaemic-hypoglycaemic clamp on day 8 after three episodes of insulin-induced hypoglycaemia (recurrent hypoglycaemia [RH]) on days 5, 6 and 7. In a subset of rats exposed to RH, NN414 infusion was terminated on day 7 to wash out NN414 before examination of counter-regulation on day 8. To determine whether continuous NN414 exposure altered K(ATP) function, we used the hypothalamic glucose-sensing GT1-7 cell line, which expresses the SUR-1-containing K(ATP) channel. RESULTS: Continuous exposure to NN414 in the setting of RH increased, rather than decreased, the glucose infusion rate (GIR), as exemplified by attenuated adrenaline (epinephrine) secretion. Termination of NN414 on day 7 with subsequent washout for 24 h partially diminished the GIR. The same duration of exposure of GT1-7 cells to NN414 substantially reduced K(ATP) conductance, which was also reversed on washout of the agonist. The suppression of K(ATP) current was not associated with reduced channel subunit mRNA or protein levels. CONCLUSIONS/INTERPRETATION: These data indicate that continuous K(ATP) activation results in suppressed CRRs to hypoglycaemia in vivo, which in vitro is associated with the reversible conversion of KATP into a stable inactive state.

AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels.

Glucose-sensing (GS) behaviour in pancreatic ß-cells is dependent on ATP-sensitive K(+) channel (KATP) activity, which is controlled by the relative levels of the KATP ligands ATP and ADP, responsible for closing and opening KATP, respectively. However, the mechanism by which ß-cells transfer energy status from mitochondria to KATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMP-activated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep KATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP5A blocked this action of AMPK. Using conditions that maximised AMPK-induced KATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that KATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane KATP by phosphotransfer cascades.

Anorexigenic and orexigenic hormone modulation of mammalian target of rapamycin complex 1 activity and the regulation of hypothalamic agouti-related protein mRNA expression.

Activation of mammalian target of rapamycin 1 (mTORC1) by nutrients, insulin and leptin leads to appetite suppression (anorexia). Contrastingly, increased AMP-activated protein kinase (AMPK) activity by ghrelin promotes appetite (orexia). However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP) mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K)- and protein kinase B (PKB)-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

Reduction in BACE1 decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice.

Insulin resistance and impaired glucose homoeostasis are important indicators of Type 2 diabetes and are early risk factors of AD (Alzheimer's disease). An essential feature of AD pathology is the presence of BACE1 (ß-site amyloid precursor protein-cleaving enzyme 1), which regulates production of toxic amyloid peptides. However, whether BACE1 also plays a role in glucose homoeostasis is presently unknown. We have used transgenic mice to analyse the effects of loss of BACE1 on body weight, and lipid and glucose homoeostasis. BACE1-/- mice are lean, with decreased adiposity, higher energy expenditure, and improved glucose disposal and peripheral insulin sensitivity than wild-type littermates. BACE1-/- mice are also protected from diet-induced obesity. BACE1-deficient skeletal muscle and liver exhibit improved insulin sensitivity. In a skeletal muscle cell line, BACE1 inhibition increased glucose uptake and enhanced insulin sensitivity. The loss of BACE1 is associated with increased levels of UCP1 (uncoupling protein 1) in BAT (brown adipose tissue) and UCP2 and UCP3 mRNA in skeletal muscle, indicative of increased uncoupled respiration and metabolic inefficiency. Thus BACE1 levels may play a critical role in glucose and lipid homoeostasis in conditions of chronic nutrient excess. Therefore strategies that ameliorate BACE1 activity may be important novel approaches for the treatment of diabetes.

Metformin increases the uptake of glucose into the gut from the circulation in high-fat diet-fed male mice, which is enhanced by a reduction in whole-body Slc2a2 expression.

OBJECTIVES: Metformin is the first line therapy recommended for type 2 diabetes. However, the precise mechanism of action remains unclear and up to a quarter of patients show some degree of intolerance to the drug, with a similar number showing poor response to treatment, limiting its effectiveness. A better understanding of the mechanism of action of metformin may improve its clinical use. SLC2A2 (GLUT2) is a transmembrane facilitated glucose transporter, with important roles in the liver, gut and pancreas. Our group previously identified single nucleotide polymorphisms in the human SLC2A2 gene, which were associated with reduced transporter expression and an improved response to metformin treatment. The aims of this study were to model Slc2a2 deficiency and measure the impact on glucose homoeostasis and metformin response in mice. METHODS: We performed extensive metabolic phenotyping and 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG)-positron emission tomography (PET) analysis of gut glucose uptake in high-fat diet-fed (HFD) mice with whole-body reduced Slc2a2 (Slc2a2+/-) and intestinal Slc2a2 KO, to assess the impact of metformin treatment. RESULTS: Slc2a2 partial deficiency had no major impact on body weight and insulin sensitivity, however mice with whole-body reduced Slc2a2 expression (Slc2a2+/-) developed an age-related decline in glucose homoeostasis (as measured by glucose tolerance test) compared to wild-type (Slc2a2+/+) littermates. Glucose uptake into the gut from the circulation was enhanced by metformin exposure in Slc2a2+/+ animals fed HFD and this action of the drug was significantly higher in Slc2a2+/- animals. However, there was no effect of specifically knocking-out Slc2a2 in the mouse intestinal epithelial cells. CONCLUSIONS: Overall, this work identifies a differential metformin response, dependent on expression of the SLC2A2 glucose transporter, and also adds to the growing evidence that metformin efficacy includes modifying glucose transport in the gut. We also describe a novel and important role for this transporter in maintaining efficient glucose homoeostasis during ageing.

Identifying the beta-site amyloid precursor protein cleaving enzyme 1 interactome through the proximity-dependent biotin identification assay.

Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a key drug target against Alzheimer's Disease however, due to its promiscuous proteolytic activity, little is known about its physiological functions. Previous studies have analysed BACE1 cleavage products to examine BACE1 interactions and determine substrates, but these studies cannot establish non-enzymatic (and potentially functional) associations. This study used the biotin identification proximity assay to establish the BACE1 interactome in healthy neuronal cells and identified interactions involved in BACE1 trafficking, post-translational modification and substrates. Furthermore, this method has identified a putative novel role for BACE1 in sex hormone signalling and haem regulation through interaction with the progesterone receptor membrane component 2 (PGRC2). Data are available via ProteomeXchange with identifier PXD021464.

Loss of AMP-activated protein kinase alpha2 subunit in mouse beta-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia.

AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic beta-cell function remains unclear. In the present study, we therefore investigated whether AMPK plays a critical function in beta-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPK alpha2 in beta-cells and a population of hypothalamic neurons (RIPCre alpha2KO mice) and RIPCre alpha2KO mice lacking AMPK alpha1 (alpha1KORIPCre alpha2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured beta-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCre alpha2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in alpha1KORIPCre alpha2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCre alpha2KO and alpha1KORIPCre alpha2KO mice, and conversely GSIS was impaired. Beta-cells lacking AMPK alpha2 or expressing a kinase-dead AMPK alpha2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCre alpha2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse beta-cell hyperpolarization, mimicking the effect of AMPK alpha2 loss. These results show that AMPK alpha2 activity is necessary to maintain normal pancreatic beta-cell glucose sensing, possibly by maintaining high beta-cell levels of UCP2.

Non-canonical Keap1-independent activation of Nrf2 in astrocytes by mild oxidative stress.

The transcription factor Nrf2 is a stress-responsive master regulator of antioxidant, detoxification and proteostasis genes. In astrocytes, Nrf2-dependent gene expression drives cell-autonomous cytoprotection and also non-cell-autonomous protection of nearby neurons, and can ameliorate pathology in several acute and chronic neurological disorders associated with oxidative stress. However, the value of astrocytic Nrf2 as a therapeutic target depends in part on whether Nrf2 activation by disease-associated oxidative stress occludes the effect of any Nrf2-activating drug. Nrf2 activation classically involves the inhibition of interactions between Nrf2's Neh2 domain and Keap1, which directs Nrf2 degradation. Keap1 inhibition is mediated by the modification of cysteine residues on Keap1, and can be triggered by electrophilic small molecules such as tBHQ. Here we show that astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling. Keap1 deficiency elevates basal Nrf2 target gene expression in astrocytes and occludes the effects of tBHQ, oxidative stress still induced strong Nrf2-dependent gene expression in Keap1-deficient astrocytes. Moreover, while tBHQ prevented protein degradation mediated via Nrf2's Neh2 domain, oxidative stress did not, consistent with a Keap1-independent mechanism. Moreover the effects of oxidative stress and tBHQ on Nrf2 target gene expression are additive, not occlusive. Mechanistically, oxidative stress enhances the transactivation potential of Nrf2's Neh5 domain in a manner dependent on its Cys-191 residue. Thus, astrocytic Nrf2 activation by oxidative stress involves Keap1-independent non-canonical signaling, meaning that further Nrf2 activation by Keap1-inhibiting drugs may be a viable therapeutic strategy.

The genetic association of the transcription factor NPAT with glycemic response to metformin involves regulation of fuel selection.

The biguanide, metformin, is the first-choice therapeutic agent for type-2 diabetes, although the mechanisms that underpin metformin clinical efficacy remain the subject of much debate, partly due to the considerable variation in patient response to metformin. Identification of poor responders by genotype could avoid unnecessary treatment and provide clues to the underlying mechanism of action. GWAS identified SNPs associated with metformin treatment success at a locus containing the NPAT (nuclear protein, ataxia-telangiectasia locus) and ATM (ataxia-telangiectasia mutated) genes. This implies that gene sequence dictates a subsequent biological function to influence metformin action. Hence, we modified expression of NPAT in immortalized cell lines, primary mouse hepatocytes and mouse tissues, and analysed the outcomes on metformin action using confocal microscopy, immunoblotting and immunocytochemistry. In addition, we characterised the metabolic phenotype of npat heterozygous knockout mice and established the metformin response following development of insulin resistance. NPAT protein was localised in the nucleus at discrete loci in several cell types, but over-expression or depletion of NPAT in immortalised cell models did not change cellular responses to biguanides. In contrast, metformin regulation of respiratory exchange ratio (RER) was completely lost in animals lacking one allele of npat. There was also a reduction in metformin correction of impaired glucose tolerance, however no other metabolic abnormalities, or response to metformin, were found in the npat heterozygous mice. In summary, we provide methodological advancements for the detection of NPAT, demonstrate that minor reductions in NPAT mRNA levels (20-40%) influence metformin regulation of RER, and propose that the association between NPAT SNPs and metformin response observed in GWAS, could be due to loss of metformin modification of cellular fuel usage.

Adipocyte integrin-linked kinase plays a key role in the development of diet-induced adipose insulin resistance in male mice.

OBJECTIVE: Increased deposition of the extracellular matrix (ECM) in adipose tissue (AT) during obesity contributes to insulin resistance. The integrin receptors transmit changes in the extracellular environment causing corresponding intracellular adaptations. Integrin-linked kinase (ILK), an adaptor protein, is a central hub for intracellular signaling of integrins. This study determined the role of ILK in adipose function and insulin resistance. METHODS: The pathogenic role of ILK in obesity and insulin resistance was studied in human adipose tissue and adipocyte-specific ILK-deficient mice (ILKlox/loxAdCre). ILKlox/loxAdCre mice together with wild-type littermates (ILKlox/lox) were fed a chow diet or 60% high-fat (HF) diet for 16 weeks. In vivo insulin sensitivity was determined by hyperinsulinemic-euglycemic clamps. RESULTS: AT ILK expression was increased by HF diet feeding in mice and increased in visceral fat of morbidly obese humans. The HF-fed ILKlox/loxAdCre mice displayed reduced fat mass and improved glucose tolerance relative to the HF-fed ILKlox/lox mice. During a hyperinsulinemic-euglycemic clamp, the HF-fed ILKlox/loxAdCre mice exhibited partially improved insulin resistance in AT. Lipolysis was suppressed to a greater extent by insulin and glucose uptake in brown AT (BAT) increased. Increased inhibition of lipolysis may have been attributed to increased vascularization in white AT, while increased glucose uptake in BAT was associated with increased Akt phosphorylation and P38/JNK dephosphorylation. Notably, AT insulin sensitivity in lean mice was not affected by ILK deletion. Moreover, reduced fat mass in the HF-fed ILKlox/loxAdCre mice may have been attributed to decreased free fatty acid uptake into adipocytes via the downregulation of CD36 gene expression. Consistent with the results in the mice, knockdown and knockout of ILK in 3T3-L1 cells decreased lipid accumulation and CD36 gene expression during adipogenesis. CONCLUSIONS: These data show that adipocyte ILK is important for regulating HF diet-mediated insulin resistance in AT in a manner consistent with AT function.