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.

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.

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.

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.

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.

Calcium Channel CaV2.3 Subunits Regulate Hepatic Glucose Production by Modulating Leptin-Induced Excitation of Arcuate Pro-opiomelanocortin Neurons.

Leptin acts on hypothalamic pro-opiomelanocortin (POMC) neurons to regulate glucose homeostasis, but the precise mechanisms remain unclear. Here, we demonstrate that leptin-induced depolarization of POMC neurons is associated with the augmentation of a voltage-gated calcium (CaV) conductance with the properties of the "R-type" channel. Knockdown of the pore-forming subunit of the R-type (CaV2.3 or Cacna1e) conductance in hypothalamic POMC neurons prevented sustained leptin-induced depolarization. In vivo POMC-specific Cacna1e knockdown increased hepatic glucose production and insulin resistance, while body weight, feeding, or leptin-induced suppression of food intake were not changed. These findings link Cacna1e function to leptin-mediated POMC neuron excitability and glucose homeostasis and may provide a target for the treatment of diabetes.

The beta secretase BACE1 regulates the expression of insulin receptor in the liver.

Insulin receptor (IR) plays a key role in the control of glucose homeostasis; however, the regulation of its cellular expression remains poorly understood. Here we show that the amount of biologically active IR is regulated by the cleavage of its ectodomain, by the ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), in a glucose concentration-dependent manner. In vivo studies demonstrate that BACE1 regulates the amount of IR and insulin signaling in the liver. During diabetes, BACE1-dependent cleavage of IR is increased and the amount of IR in the liver is reduced, whereas infusion of a BACE1 inhibitor partially restores liver IR. We suggest the potential use of BACE1 inhibitors to enhance insulin signaling during diabetes. Additionally, we show that plasma levels of cleaved IR reflect IR isoform A expression levels in liver tumors, which prompts us to propose that the measurement of circulating cleaved IR may assist hepatic cancer detection and management.

Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

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.

The BACE1 product sAPPß induces ER stress and inflammation and impairs insulin signaling.

OBJECTIVE: ß-secretase/ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a key enzyme involved in Alzheimer's disease that has recently been implicated in insulin-independent glucose uptake in myotubes. However, it is presently unknown whether BACE1 and the product of its activity, soluble APPß (sAPPß), contribute to lipid-induced inflammation and insulin resistance in skeletal muscle cells. MATERIALS/METHODS: Studies were conducted in mouse C2C12 myotubes, skeletal muscle from Bace1-/-mice and mice treated with sAPPß and adipose tissue and plasma from obese and type 2 diabetic patients. RESULTS: We show that BACE1 inhibition or knockdown attenuates palmitate-induced endoplasmic reticulum (ER) stress, inflammation, and insulin resistance and prevents the reduction in Peroxisome Proliferator-Activated Receptor γ Co-activator 1α (PGC-1α) and fatty acid oxidation caused by palmitate in myotubes. The effects of palmitate on ER stress, inflammation, insulin resistance, PGC-1α down-regulation, and fatty acid oxidation were mimicked by soluble APPß in vitro. BACE1 expression was increased in subcutaneous adipose tissue of obese and type 2 diabetic patients and this was accompanied by a decrease in PGC-1α mRNA levels and by an increase in sAPPß plasma levels of obese type 2 diabetic patients compared to obese non-diabetic subjects. Acute sAPPß administration to mice reduced PGC-1α levels and increased inflammation in skeletal muscle and decreased insulin sensitivity. CONCLUSIONS: Collectively, these findings indicate that the BACE1 product sAPPß is a key determinant in ER stress, inflammation and insulin resistance in skeletal muscle and gluconeogenesis in liver.

Bace1-dependent amyloid processing regulates hypothalamic leptin sensitivity in obese mice.

Obesity places an enormous medical and economic burden on society. The principal driver appears to be central leptin resistance with hyperleptinemia. Accordingly, a compound that reverses or prevents leptin resistance should promote weight normalisation and improve glucose homeostasis. The protease Bace1 drives beta amyloid (Aß) production with obesity elevating hypothalamic Bace1 activity and Aß1-42 production. Pharmacological inhibition of Bace1 reduces body weight, improves glucose homeostasis and lowers plasma leptin in diet-induced obese (DIO) mice. These actions are not apparent in ob/ob or db/db mice, indicating the requirement for functional leptin signalling. Decreasing Bace1 activity normalises hypothalamic inflammation, lowers PTP1B and SOCS3 and restores hypothalamic leptin sensitivity and pSTAT3 response in obese mice, but does not affect leptin sensitivity in lean mice. Raising central Aß1-42 levels in the early stage of DIO increases hypothalamic basal pSTAT3 and reduces the amplitude of the leptin pSTAT3 signal without increased inflammation. Thus, elevated Aß1-42 promotes hypothalamic leptin resistance, which is associated with diminished whole-body sensitivity to exogenous leptin and exacerbated body weight gain in high fat fed mice. These results indicate that Bace1 inhibitors, currently in clinical trials for Alzheimer's disease, may be useful agents for the treatment of obesity and associated diabetes.

High-Intensity Exercise as a Dishabituating Stimulus Restores Counterregulatory Responses in Recurrently Hypoglycemic Rodents.

Hypoglycemia is a major adverse effect of insulin therapy for people with type 1 diabetes (T1D). Profound defects in the normal counterregulatory response to hypoglycemia explain the frequency of hypoglycemia occurrence in T1D. Defective counterregulation results to a large extent from prior exposure to hypoglycemia per se, leading to a condition called impaired awareness of hypoglycemia (IAH), the cause of which is unknown. In the current study, we investigate the hypothesis that IAH develops through a special type of adaptive memory referred to as habituation. To test this hypothesis, we used a novel intense stimulus (high-intensity exercise) to demonstrate two classic features of a habituated response, namely dishabituation and response recovery. We demonstrate that after recurrent hypoglycemia the introduction of a novel dishabituating stimulus (a single burst of high-intensity exercise) in male Sprague-Dawley rats restores the defective hypoglycemia counterregulatory response. In addition, the rats showed an enhanced response to the novel stimulus (response recovery). We make the further observation using proteomic analysis of hypothalamic extracts that high-intensity exercise in recurrently hypoglycemic rats increases levels of a number of proteins linked with brain-derived neurotrophic factor signaling. These findings may lead to novel therapeutic approaches for individuals with T1D and IAH.

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.

Oleate induces KATP channel-dependent hyperpolarization in mouse hypothalamic glucose-excited neurons without altering cellular energy charge.

The unsaturated fatty acid, oleate exhibits anorexigenic properties reducing food intake and hepatic glucose output. However, its mechanism of action in the hypothalamus has not been fully determined. This study investigated the effects of oleate and glucose on GT1-7 mouse hypothalamic cells (a model of glucose-excited (GE) neurons) and mouse arcuate nucleus (ARC) neurons. Whole-cell and perforated patch-clamp recordings, immunoblotting and cell energy status measures were used to investigate oleate- and glucose-sensing properties of mouse hypothalamic neurons. Oleate or lowered glucose concentration caused hyperpolarization and inhibition of firing of GT1-7 cells by the activation of ATP-sensitive K+ channels (KATP). This effect of oleate was not dependent on fatty acid oxidation or raised AMP-activated protein kinase activity or prevented by the presence of the UCP2 inhibitor genipin. Oleate did not alter intracellular calcium, indicating that CD36/fatty acid translocase may not play a role. However, oleate activation of KATP may require ATP metabolism. The short-chain fatty acid octanoate was unable to replicate the actions of oleate on GT1-7 cells. Although oleate decreased GT1-7 cell mitochondrial membrane potential there was no change in total cellular ATP or ATP/ADP ratios. Perforated patch and whole-cell recordings from mouse hypothalamic slices demonstrated that oleate hyperpolarized a subpopulation of ARC GE neurons by KATP activation. Additionally, in a separate small population of ARC neurons, oleate application or lowered glucose concentration caused membrane depolarization. In conclusion, oleate induces KATP-dependent hyperpolarization and inhibition of firing of a subgroup of GE hypothalamic neurons without altering cellular energy charge.

Chronic exposure to KATP channel openers results in attenuated glucose sensing in hypothalamic GT1-7 neurons.

Individuals with Type 1 diabetes (T1D) are often exposed to recurrent episodes of hypoglycaemia. This reduces hormonal and behavioural responses that normally counteract low glucose in order to maintain glucose homeostasis, with altered responsiveness of glucose sensing hypothalamic neurons implicated. Although the molecular mechanisms are unknown, pharmacological studies implicate hypothalamic ATP-sensitive potassium channel (KATP) activity, with KATP openers (KCOs) amplifying, through cell hyperpolarization, the response to hypoglycaemia. Although initial findings, using acute hypothalamic KCO delivery, in rats were promising, chronic exposure to the KCO NN414 worsened the responses to subsequent hypoglycaemic challenge. To investigate this further we used GT1-7 cells to explore how NN414 affected glucose-sensing behaviour, the metabolic response of cells to hypoglycaemia and KATP activity. GT1-7 cells exposed to 3 or 24 h NN414 exhibited an attenuated hyperpolarization to subsequent hypoglycaemic challenge or NN414, which correlated with diminished KATP activity. The reduced sensitivity to hypoglycaemia was apparent 24 h after NN414 removal, even though intrinsic KATP activity recovered. The NN414-modified glucose responsiveness was not associated with adaptations in glucose uptake, metabolism or oxidation. KATP inactivation by NN414 was prevented by the concurrent presence of tolbutamide, which maintains KATP closure. Single channel recordings indicate that NN414 alters KATP intrinsic gating inducing a stable closed or inactivated state. These data indicate that exposure of hypothalamic glucose sensing cells to chronic NN414 drives a sustained conformational change to KATP, probably by binding to SUR1, that results in loss of channel sensitivity to intrinsic metabolic factors such as MgADP and small molecule agonists.

Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63 ubiquitin chain formation.

Dimethyl fumarate (DMF) possesses anti-inflammatory properties and is approved for the treatment of psoriasis and multiple sclerosis. While clinically effective, its molecular target has remained elusive - although it is known to activate anti-oxidant pathways. We find that DMF inhibits pro-inflammatory cytokine production in response to TLR agonists independently of the Nrf2-Keap1 anti-oxidant pathway. Instead we show that DMF can inhibit the E2 conjugating enzymes involved in K63 and M1 polyubiquitin chain formation both in vitro and in cells. The formation of K63 and M1 chains is required to link TLR activation to downstream signaling, and consistent with the block in K63 and/or M1 chain formation, DMF inhibits NFκB and ERK1/2 activation, resulting in a loss of pro-inflammatory cytokine production. Together these results reveal a new molecular target for DMF and show that a clinically approved drug inhibits M1 and K63 chain formation in TLR induced signaling complexes. Selective targeting of E2s may therefore be a viable strategy for autoimmunity.

Nrf2-Mediated Neuroprotection Against Recurrent Hypoglycemia Is Insufficient to Prevent Cognitive Impairment in a Rodent Model of Type 1 Diabetes.

It remains uncertain whether recurrent nonsevere hypoglycemia (Hypo) results in long-term cognitive impairment in type 1 diabetes (T1D). This study tested the hypothesis that specifically in the T1D state, Hypo leads to cognitive impairment via a pathological response to oxidative stress. Wild-type (Control) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) null mice were studied. Eight groups of mice (Control and Nrf2(-/-) ± T1D and ± Hypo) were subject to recurrent, twice-weekly, insulin or saline injections over 4 weeks, after which cognitive function was assessed and brain tissue analyzed. Recurrent moderate hypoglycemia in T1D, but not Control, mice significantly impaired cognitive performance, and this was associated with hippocampal oxidative damage and inflammation despite an enhanced expression of Nrf2 and its target genes Hmox1 and Nqo1 In Nrf2(-/-) mice, both T1D and Hypo independently resulted in impaired cognitive performance, and this was associated with oxidative cell damage and marked inflammation. Together, these data suggest that Hypo induces an Nrf2-dependent antioxidant response in the hippocampus, which counteracts oxidative damage. However, in T1D, this neuroprotective mechanism is insufficient to prevent neuronal oxidative damage, resulting in chronic deficits in working and long-term memory.