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Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver–brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases.

Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked β-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.

Summary: Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1β (IL-1β) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases β-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1β in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout. : NLRP3 inflammasome activation in macrophages and neutrophils drives painful inflammation during gout. Goldberg et al. report that ketogenic diet prevents systemic inflammation and joint damage in a rat model of gouty flare. Mechanistically, the ketone body β-hydroxybutyrate, the most abundant ketone in vivo, inhibits NLRP3/caspase-1-dependent IL-1β secretion from neutrophils. Keywords: NLRP3 inflammasome, gout, β-hydroxybutyrate, inflammation, aging, neutrophil, IL-1

Mitophagy can selectively remove damaged toxic mitochondria, protecting a cell from apoptosis. The molecular spatial–temporal mechanisms governing autophagosomal selection of reactive oxygen species (ROS)‐damaged mitochondria, particularly in a platelet (no genomic DNA for transcriptional regulation), remain unclear. We now report that the mitochondrial matrix protein MsrB2 plays an important role in switching on mitophagy by reducing Parkin methionine oxidation (MetO), and transducing mitophagy through ubiquitination by Parkin and interacting with LC3. This biochemical signaling only occurs at damaged mitochondria where MsrB2 is released from the mitochondrial matrix. MsrB2 platelet‐specific knockout and in vivo peptide inhibition of the MsrB2/LC3 interaction lead to reduced mitophagy and increased platelet apoptosis. Pathophysiological importance is highlighted in human subjects, where increased MsrB2 expression in diabetes mellitus leads to increased platelet mitophagy, and in platelets from Parkinson's disease patients, where reduced MsrB2 expression is associated with reduced mitophagy. Moreover, Parkin mutations at Met192 are associated with Parkinson's disease, highlighting the structural sensitivity at the Met192 position. Release of the enzyme MsrB2 from damaged mitochondria, initiating autophagosome formation, represents a novel regulatory mechanism for oxidative stress‐induced mitophagy.

Null mutations in for pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, are the cause of osteogenesis imperfecta (OI) type VI. The PEDF-knockout (KO) mouse captures crucial elements of the human disease, including diminished bone mineralization and propensity to fracture. Our group and others have demonstrated that PEDF directs human mesenchymal stem cell (hMSC) commitment to the osteoblast lineage and modulates Wnt/β-catenin signaling, a major regulator of bone development; however, the ability of PEDF to restore bone mass in a mouse model of OI type VI has not been determined. In this study, PEDF delivery increased trabecular bone volume/total volume by 52% in 6-mo-old PEDF-KO mice but not in wild-type mice. In young (19-d-old) PEDF-KO mice, PEDF restoration increased bone volume fraction by 35% and enhanced biomechanical parameters of bone plasticity. A Wnt-green fluorescent protein reporter demonstrated dynamic changes in Wnt/β-catenin signaling characterized by early activation and marked suppression during terminal differentiation of hMSCs. Continuous Wnt3a exposure impeded mineralization of hMSCs, whereas the combination of Wnt3a and PEDF potentiated mineralization. Interrogation of the PEDF sequence identified a conserved motif found in other Wnt modulators, such as the dickkopf proteins. Mutation of a single amino acid on a 34-mer PEDF peptide increased mineralization of hMSC cultures compared with the native peptide sequence. These results indicate that PEDF counters Wnt signaling to allow for osteoblast differentiation and provides a mechanistic insight into how the PEDF null state results in OI type VI.-Belinsky, G. S., Sreekumar, B., Andrejecsk, J. W., Saltzman, W. M., Gong, J., Herzog, R. I., Lin, S., Horsley, V., Carpenter, T. O., Chung, C. Pigment epithelium-derived factor restoration increases bone mass and improves bone plasticity in a model of osteogenesis imperfecta type VI via Wnt3a blockade.

Our objective was to evaluate whether excessive brain glycogen deposition might follow episodes of acute hypoglycemia (AH) and thus play a role in the hypoglycemia-associated autonomic failure seen in diabetic patients receiving intensive insulin treatment. We determined brain glucose and glycogen recovery kinetics after AH and recurrent hypoglycemia (RH), an established animal model of counterregulatory failure. A single bout of insulin-induced AH or RH for 3 consecutive days was used to deplete brain glucose and glycogen stores in rats. After microwave fixation and glycogen extraction, regional recovery kinetics in the brain was determined using a biochemical assay. Both AH and RH treatments reduced glycogen levels in the cerebellum, cortex, and hypothalamus from control levels of 7.78 +/- 0.55, 5.4 +/- 0.38, and 4.45 +/- 0.37 micromol/g, respectively, to approximately 50% corresponding to a net glycogen utilization rate between 0.6 and 1.2 micromol/g.h. After hypoglycemia, glycogen levels returned to baseline within 6 h in both the AH and the RH group. However, recovery of brain glycogen tended to be faster in rats exposed to RH. This effect followed more rapid recovery of brain glucose levels in the RH group, despite similar blood glucose levels in both groups. There was no statistically significant increase above baseline glycogen levels in either group. In particular, brain glycogen was not increased 24 h after the last of recurrent episodes of hypoglycemia, when a significant counterregulatory defect could be documented during a hyperinsulinemic hypoglycemic clamp study. We conclude that glycogen supercompensation is not a major contributory factor to the pathogenesis of hypoglycemia-associated autonomic failure.

Voltage-gated sodium channels can be characterized by their sensitivity to inhibitors. Na(v)1.5 is sensitive to block by cadmium and extracellular QX-314, but relatively insensitive to tetrodotoxin and saxitoxin. Na(v)1.4 is tetrodotoxin- and saxitoxin-sensitive but resistant to cadmium and extracellular QX-314. Na(v)1.8 and Na(v)1.9 generate slowly inactivating (I(TTXr-Slow)) and persistent (I(TTXr-Per)) currents in sensory neurons that are tetrodotoxin-resistant. Tetrodotoxin sensitivity is largely determined by the identity of a single residue; tyrosine 401 in Na(v)1.4, cysteine 374 in Na(v)1.5 and serine 356 and 355 in Na(v)1.8 and Na(v)1.9. We asked whether Na(v)1.8 and Na(v)1.9 share other pharmacological properties as a result of this serine residue. I(TTXr-Slow) and I(TTXr-Per) were saxitoxin-resistant and resistant to internal QX-314. I(TTXr-Slow) was also resistant to external QX-314 and displayed a approximately fourfold higher sensitivity than I(TTXr-Per) to cadmium. The impact of the serine residue was investigated by replacing tyrosine 401 in Na(v)1.4 with serine (Y401S) or cysteine (Y401C). Both mutants were resistant to tetrodotoxin and saxitoxin. Whereas Na(v)1.4-Y401C displayed an increased sensitivity to cadmium and extracellular QX-314, the serine substitution did not alter the sensitivity of Na(v)1.4 to cadmium or QX-314. Our data indicates that while the serine residue determines the sensitivity of I(TTXr-Slow) and I(TTXr-Per) to tetrodotoxin and saxitoxin, it does not determine their insensitivity to QX-314 or their differential sensitivities to cadmium.

The Nav1.6 voltage-gated sodium channel has been implicated in the generation of resurgent currents in cerebellar Purkinje neurons. Our data show that resurgent sodium currents are produced by some large diameter dorsal root ganglion (DRG) neurons from wild-type mice, but not from Nav1.6-null mice; small DRG neurons do not produce resurgent currents. Many, but not all, DRG neurons transfected with Nav1.6 produce resurgent currents. These results demonstrate for the first time the intrinsic ability of Nav1.6 to produce a resurgent current, and also show that cell background is critical in permitting the generation of these currents.

While large, myelinated dorsal root ganglion (DRG) neurons are capable of firing at high frequencies, small unmyelinated DRG neurons typically display much lower maximum firing frequencies. However, the molecular basis for this difference has not been delineated. Because the sodium currents in large DRG neurons exhibit rapid repriming (recovery from inactivation) kinetics and the sodium currents in small DRG neurons exhibit predominantly slow repriming kinetics, it has been proposed that differences in sodium channels might contribute to the determination of repetitive firing properties in DRG neurons. A recent study demonstrated that Nav1.7 expression is negatively correlated with conduction velocity and DRG cell size, while the Nav1.6 voltage-gated sodium channel has been implicated as the predominant isoform present at nodes of Ranvier of myelinated fibres. Therefore we characterized and compared the functional properties, including repriming, of recombinant Nav1.6 and Nav1.7 channels expressed in mouse DRG neurons. Both Nav1.6 and Nav1.7 channels generated fast-activating and fast-inactivating currents. However recovery from inactivation was significantly faster (approximately 5-fold at -70 mV) for Nav1.6 currents than for Nav1.7 currents. The recovery from inactivation of Nav1.6 channels was also much faster than that of native tetrodotoxin-sensitive sodium currents recorded from small spinal sensory neurons, but similar to that of tetrodotoxin-sensitive sodium currents recorded from large spinal sensory neurons. Development of closed-state inactivation was also much faster for Nav1.6 currents than for Nav1.7 currents. Our results indicate that the firing properties of DRG neurons can be tuned by regulating expression of different sodium channel isoforms that have distinct repriming and closed-state inactivation kinetics.

An endogenous pentapeptide (Gln-Tyr-Asn-Ala-Asp; QYNAD) that is present at elevated levels in human CSF from patients with demyelinating diseases has been reported to block voltage-gated sodium channels at low (10 micro M) concentrations. Objective : Because of the potential importance of sodium channel blocking activity in demyelinating disorders, this study attempted to determine the sensitivity to QYNAD of different sodium channel subtypes, including Na(v)1.6, the major sodium channel at nodes of Ranvier, and Na(v)1.2, which is expressed in axons with abnormal myelin.Sodium channel function was assayed using patch-clamp recordings, both in heterologous expression systems and in intact neurons.QYNAD synthesized in 10 different batches by four different facilities failed to block sodium currents, even at concentrations as high as 500 micro M (50-fold higher than the blocking concentration originally reported). QYNAD had no effect on the currents produced by recombinant Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.7 sodium channels or on the sodium currents that are produced by native channels in adult hippocampal or dorsal root ganglion neurons. QYNAD did not interfere with conduction in the optic nerve, a myelinated fiber tract that is often affected in MS.These experiments do not show any sodium channel blocking effect of QYNAD. The conclusion that QYNAD contributes to the pathophysiology of inflammatory neurologic disorders by blocking voltage-gated sodium channels should therefore be viewed with caution.

An elevated level of von Willebrand factor (VWF) in diabetic patients is associated with increased risk of thrombotic cardiovascular events. The underlying mechanism of how VWF expression is upregulated in diabetes mellitus is poorly understood. We now report that hyperglycemia-induced repression of microRNA-24 (miR-24) increases VWF expression and secretion in diabetes mellitus. In diabetic patients and diabetic mouse models (streptozotocin/high-fat diet-induced and db/db mice), miR-24 is reduced in both tissues and plasma. Knockdown of miR-24 in mice leads to increased VWF mRNA and protein levels and enhanced platelet tethering (spontaneous thrombosis). miR-24 tightly controls VWF levels through pleiotropic effects, including direct binding to the 3′ untranslated region of VWF and targeting FURIN and the histamine H1 receptor, known regulators of VWF processing and secretion in endothelial cells. We present a novel mechanism for miR-24 downregulation through hyperglycemia-induced activation of aldose reductase, reactive oxygen species, and c-Myc. These findings support a critical role for hyperglycemic repression of miR-24 in VWF-induced pathology. miR-24 represents a novel therapeutic target to prevent adverse thrombotic events in patients with diabetes mellitus.

Small dorsal root ganglion (DRG) neurons, which include nociceptors, express multiple voltage-gated sodium currents. In addition to a classical fast inactivating tetrodotoxin-sensitive (TTX-S) sodium current, many of these cells express a TTX-resistant (TTX-R) sodium current that activates near −70 mV and is persistent at negative potentials. To investigate the possible contributions of this TTX-R persistent (TTX-RP) current to neuronal excitability, we carried out computer simulations using the Neuron program with TTX-S and -RP currents, fit by the Hodgkin-Huxley model, that closely matched the currents recorded from small DRG neurons. In contrast to fast TTX-S current, which was well fit using a m3h model, the persistent TTX-R current was not well fit by an m3h model and was better fit using an mh model. The persistent TTX-R current had a strong influence on resting potential, shifting it from −70 to −49.1 mV. Inclusion of an ultra-slow inactivation gate in the persistent current model reduced the potential shift only slightly, to −56.6 mV. The persistent TTX-R current also enhanced the response to depolarizing inputs that were subthreshold for spike electrogenesis. In addition, the presence of persistent TTX-R current predisposed the cell to anode break excitation. These results suggest that, while the persistent TTX-R current is not a major contributor to the rapid depolarizing phase of the action potential, it contributes to setting the electrogenic properties of small DRG neurons by modulating their resting potentials and response to subthreshold stimuli.

OBJECTIVE The objective of this study was to characterize the changes in brain metabolism caused by antecedent recurrent hypoglycemia under euglycemic and hypoglycemic conditions in a rat model and to test the hypothesis that recurrent hypoglycemia changes the brain's capacity to utilize different energy substrates. RESEARCH DESIGN AND METHODS Rats exposed to recurrent insulin-induced hypoglycemia for 3 days (3dRH rats) and untreated controls were subject to the following protocols: [2-13C]acetate infusion under euglycemic conditions (n = 8), [1-13C]glucose and unlabeled acetate coinfusion under euglycemic conditions (n = 8), and [2-13C]acetate infusion during a hyperinsulinemic-hypoglycemic clamp (n = 8). In vivo nuclear magnetic resonance spectroscopy was used to monitor the rise of13C-labeling in brain metabolites for the calculation of brain metabolic fluxes using a neuron-astrocyte model. RESULTS At euglycemia, antecedent recurrent hypoglycemia increased whole-brain glucose metabolism by 43 ± 4% (P < 0.01 vs. controls), largely due to higher glucose utilization in neurons. Although acetate metabolism remained the same, control and 3dRH animals showed a distinctly different response to acute hypoglycemia: controls decreased pyruvate dehydrogenase (PDH) flux in astrocytes by 64 ± 20% (P = 0.01), whereas it increased by 37 ± 3% in neurons (P = 0.01). The 3dRH animals decreased PDH flux in both compartments (−75 ± 20% in astrocytes, P < 0.001, and −36 ± 4% in neurons, P = 0.005). Thus, acute hypoglycemia reduced total brain tricarboxylic acid cycle activity in 3dRH animals (−37 ± 4%, P = 0.001), but not in controls. CONCLUSIONS Our findings suggest that after antecedent hypoglycemia, glucose utilization is increased at euglycemia and decreased after acute hypoglycemia, which was not the case in controls. These findings may help to identify better methods of preserving brain function and reducing injury during acute hypoglycemia.

OBJECTIVE—We have previously demonstrated that modulation of γ-aminobutyric acid (GABA) inhibitory tone in the ventromedial hypothalamus (VMH), an important glucose-sensing region in the brain, modulates the magnitude of glucagon and sympathoadrenal responses to hypoglycemia. In the current study, we examined whether increased VMH GABAergic tone may contribute to suppression of counterregulatory responses after recurrent hypoglycemia. RESEARCH DESIGN AND METHODS—To test this hypothesis, we quantified expression of the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in the VMH of control and recurrently hypoglycemic rats. Subsequently, we used microdialysis and microinjection techniques to assess changes in VMH GABA levels and the effects of GABAA receptor blockade on counterregulatory responses to a standardized hypoglycemic stimulus. RESULTS—Quantitative RT-PCR and immunoblots in recurrently hypoglycemic animals revealed that GAD65 mRNA and protein were increased 33 and 580%, respectively. Basal VMH GABA concentrations were more than threefold higher in recurrently hypoglycemic animals. Furthermore, whereas VMH GABA levels decreased in both control and recurrently hypoglycemic animals with the onset of hypoglycemia, the fall was not significant in recurrently hypoglycemic rats. During hypoglycemia, recurrently hypoglycemic rats exhibited a 49–63% reduction in glucagon and epinephrine release. These changes were reversed by delivery of a GABAA receptor antagonist to the VMH. CONCLUSIONS—Our data suggest that recurrent hypoglycemia increases GABAergic inhibitory tone in the VMH and that this, in turn, suppresses glucagon and sympathoadrenal responses to subsequent bouts of acute hypoglycemia. Thus, hypoglycemia-associated autonomic failure may be due in part to a relative excess of the inhibitory neurotransmitter, GABA, within the VMH.

Modulation of voltage-gated sodium channels (VGSC) can have a major impact on cell excitability. Analysis of calmodulin (CaM) binding to GST-fusion proteins containing the C-terminal domains of Nav1.1-Nav1.9 indicates that some of the tetrodotoxin-sensitive VGSC isoforms, including NaV1.4 and NaV1.6, are able to bind CaM in a calcium-independent manner. Here we demonstrate that association with CaM is important for functional expression of NaV1.4 and NaV1.6 VGSCs. Disrupting the interaction between CaM and the C terminus of NaV1.4 and NaV1.6 channels reduced current amplitude by 99 and 62%, respectively. Overexpression of CaM increased the current generated by Nav1.4 and Nav1.6 C-terminal mutant constructs that exhibited intermediate current densities and intermediate binding affinities for CaM, demonstrating that this effect on current density was directly dependent on the ability of the C terminus to bind CaM. In addition to the effects on current density, calmodulin also was able to modulate the inactivation kinetics of Nav1.6, but not Nav1.4, currents in a calcium-dependent manner. Our data demonstrate that CaM can regulate the properties of VGSCs via calcium-dependent and calcium-independent mechanisms and suggest that modulation of neuronal sodium channels may play a role in calcium-dependent neuronal plasticity.

Although rat brain Nav1.3 voltage-gated sodium channels have been expressed and studied in Xenopus oocytes, these channels have not been studied after their expression in mammalian cells. We characterized the properties of the rat brain Nav1.3 sodium channels expressed in human embryonic kidney (HEK) 293 cells. Nav1.3 channels generated fast-activating and fast-inactivating currents. Recovery from inactivation was relatively rapid at negative potentials (-80 mV) but was slow at more positive potentials. Development of closed-state inactivation was slow, and, as predicted on this basis, Nav1.3 channels generated large ramp currents in response to slow depolarizations. Coexpression of beta3 subunits had small but significant effects on the kinetic and voltage-dependent properties of Nav1.3 currents in HEK 293 cells, but coexpression of beta1 and beta2 subunits had little or no effect on Nav1.3 properties. Nav1.3 channels, mutated to be tetrodotoxin-resistant (TTX-R), were expressed in SNS-null dorsal root ganglion (DRG) neurons via biolistics and were compared with the same construct expressed in HEK 293 cells. The voltage dependence of steady-state inactivation was approximately 7 mV more depolarized in SNS-null DRG neurons, demonstrating the importance of background cell type in determining physiological properties. Moreover, consistent with the idea that cellular factors can modulate the properties of Nav1.3, the repriming kinetics were twofold faster in the neurons than in the HEK 293 cells. The rapid repriming of Nav1.3 suggests that it contributes to the acceleration of repriming of TTX-sensitive (TTX-S) sodium currents that are seen after peripheral axotomy of DRG neurons. The relatively rapid recovery from inactivation and the slow closed-state inactivation kinetics of Nav1.3 channels suggest that neurons expressing Nav1.3 may exhibit a reduced threshold and/or a relatively high frequency of firing.

To develop a vaccination approach for prevention of type 1 diabetes (T1D) that selectively attenuates self-reactive T-cells targeting specific autoantigens, we selected phage-displayed single chain antigen receptor libraries for clones binding to a complex of the NOD classII MHC I-A(g7) and epitopes derived from the islet autoantigen RegII. Libraries were generated from B-cell receptor repertoires of classII-mismatched mice immunized with RegII-pulsed NOD antigen presenting cells or from T-cell receptor repertoires in pancreatic lymph nodes of NOD mice. Both approaches yielded clones recognizing a RegII-derived epitope in the context of I-A(g7), which activated autoreactive CD4(+) T-cells. A receptor with different specificity was obtained by converting the BDC2.5 TCR into single chain form. B- but not T-cells from donors vaccinated with the clones transferred protection from diabetes to NOD-SCID recipients if the specificity of the diabetes inducer cell and the single chain receptor were matched. B-cells and antibodies from donors vaccinated with the BDC2.5 single chain receptor induced a state of profound anergy in T-cells of BDC2.5 TCR transgenic NOD recipients while B-cells from donors vaccinated with a single chain receptor specific for I-A(g7) RegII peptide complexes induced only partial non-responsiveness. Vaccination of normal NOD mice with receptors recognizing I-A(g7) RegII peptide complexes or with the BDC2.5 single chain receptor delayed onset of T1D. Thus anti-idiotypic vaccination can be successfully applied to T1D with vaccines either generated from self-reactive T-cell clones or derived from antigen receptor libraries.