Lipocalin-2 Deficiency Impairs Thermogenesis and Potentiates Diet-Induced Insulin Resistance in Mice

OBJECTIVE

Lipocalin (LCN) 2 belongs to the lipocalin subfamily of low–molecular mass–secreted proteins that bind small hydrophobic molecules. LCN2 has been recently characterized as an adipose-derived cytokine, and its expression is upregulated in adipose tissue in genetically obese rodents. The objective of this study was to investigate the role of LCN2 in diet-induced insulin resistance and metabolic homeostasis in vivo.

RESEARCH DESIGN AND METHODS

Systemic insulin sensitivity, adaptive thermogenesis, and serum metabolic and lipid profile were assessed in LCN2-deficient mice fed a high-fat diet (HFD) or regular chow diet.

RESULTS

The molecular disruption of LCN2 in mice resulted in significantly potentiated diet-induced obesity, dyslipidemia, fatty liver disease, and insulin resistance. LCN2^−/− mice exhibit impaired adaptive thermogenesis and cold intolerance. Gene expression patterns in white and brown adipose tissue, liver, and muscle indicate that LCN2^−/− mice have increased hepatic gluconeogenesis, decreased mitochondrial oxidative capacity, impaired lipid metabolism, and increased inflammatory state under the HFD condition.

CONCLUSIONS

LCN2 has a novel role in adaptive thermoregulation and diet-induced insulin resistance.Obesity is a major risk for developing insulin resistance, a hallmark of type 2 diabetes and other metabolic complications such as fatty liver, dyslipidemia, and atherosclerosis. Adipose tissue plays a central role in body weight homeostasis, inflammation, and insulin resistance via regulating lipid metabolism/storage and releasing a range of adipokines/cytokines (1–4). Adipose tissue in a variety of insulin-resistant states has been characterized by dysregulated lipid metabolism and altered production of adipokines/cytokines that, in sum, are important contributors to systemic inflammation and related metabolic disorders.Lipocalin (LCN) 2 (also known as neutrophil gelatinase–associated lipocalin [NGAL]), a lipocalin subfamily member, has been recently identified by our group and others (5,6) as an adipose-derived cytokine. LCN2 is a 25-kDa secreted protein initially identified from human neutrophils (7,8) and other immune cells and tissues that are exposed to microorganisms in the respiratory and gastrointestinal tract and is present abundantly in the circulation (9). Interestingly, lipocalins have structural similarity with fatty acid binding proteins (FABPs), and both are members of the multigene family of up and down β-barrel proteins (10). Both the intracellular FABPs and the extracellular lipocalins have a clearly defined β-barrel motif that forms either an interior cavity (FABP) or a deep pit (lipocalins) that constitutes the lipid binding domain (10). The extracellular lipocalins such as LCN2, retinol binding protein (RBP) 4, and α₂-microglobulin use a series of β-strands to form a globular domain with a deep depression resembling the calyx of a flower. Because of the unique structure, the lipocalins function as efficient transporters for a number of different hydrophobic ligands in extracellular milieus, including a variety of retinoids, fatty acids, biliverdin, pheromones, porphyrins, odorants, steroids, and iron. RBP4, one of the extracellular lipocalins, affects glucose metabolism and insulin sensitivity (11).Previous studies have demonstrated that LCN2 gene expression is upregulated in adipose tissue and liver of genetically obese animals (6). Rosiglitazone administration significantly reduces LCN2 expression in adipose tissue in obese animals (6), suggesting that the protein may function as a proinflammatory factor. Unexpectedly, the addition of LCN2 protein to the culture media of adipocytes and macrophages leads to the suppression of tumor necrosis factor (TNF)α- and lipopolysaccharide-induced cytokine/chemokine production, indicating an anti-inflammatory function (6). Most strikingly, LCN2 appears to protect against TNFα-induced insulin resistance in adipocytes. Unlike RBP4, increased production of LCN2 in obesity may be a protective mechanism against inflammation and insulin resistance.To evaluate this hypothesis, we assessed the metabolic and regulatory consequences of LCN2 deficiency. Herein, we show that the ablation of LCN2 profoundly impairs adaptive thermogenesis and exacerbates high-fat diet (HFD)- or age-induced insulin resistance and glucose homeostasis. LCN2-deficient mice have increased hepatic gluconeogenesis and inflammatory state and exhibit a cold sensitive phenotype.     

Tetraethylammonium-Induced Synaptic Plasticity in Rat Neocortex

Recordings were obtained from neurons in layer II/III of slicesof rat frontal cortex maintained in vitro. We investigated whetherbrief application of the potassium channel blocker tetraethylammonium(TEA), which induces a novel form of synaptic plasticity inthe CA1 region of the hippocampus referred to as LTP_k evokessimilar responses in neocortex. Consistent with previous findings,TEA produced a persistent enhancement of excitatory transmission,which was independent of NMDA receptor activation but requiredthe activation of nifedipine-sensitive voltage-dependent Ca²⁺channels (VDCC), presumably the L-type. We also observed a persistentenhancement of presumptive Cl^–-dependent GABA_A receptor-mediatedtransmission. Enhancement of excitatory and inhibitory synaptictransmission did not require activation of synapses with electricalstimulation during TEA application. The enhancement of excitatory,but not inhibitory synaptic transmission, was blocked when theCa²⁺ chelator 1,2-bis(2-aminophenoxy)-ethane N,N,N',N'-tetraaceticacid (BAPTA) was included in the recording electrode. Undervoltage clamp conditions that minimized the activation of L-typechannels robust enhancement of both excitatory and inhibitorytransmission was still observed. No enhancement of excitatorysynaptic transmission was observed in the presence of NiCl₂,a putative T-type channel blocker. The possible involvementof kinase activation was studied by including the non-specificand competitive kinase inhibitor (±)-1-(5-isoquinolinesulfonyl)-2-methylpiperazinedihydrochloride (H-7) in the patch pipette. H-7 retarded thetime course and reduced the magnitude of the enhancement ofexcitatory transmission. These results suggest that TEA-inducedenhancement of excitatory transmission in the neocortex requiresentry of Ca²⁺ into the postsynaptic neuron via VDCCs and possiblythe activation of a kinase.     

Amnestic Concentrations of Sevoflurane Inhibit Synaptic Plasticity of Hippocampal CA1 Neurons through [gamma]-Aminobutyric Acid-mediated Mechanisms

Background: The cellular mechanisms of anesthetic-induced amnesia are still poorly understood. The current study examined sevoflurane at various concentrations in the CA1 region of rat hippocampal slices for effects on excitatory synaptic transmission and on long-term potentiation (LTP), as a possible mechanism contributing to anesthetic-induced loss of recall.

Methods: Population spikes and field excitatory postsynaptic potentials were recorded using extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic. LTP was induced using tetanic stimulation (100 Hz, 1 s). Sevoflurane at concentrations from amnestic (0.04 mm) to clinical concentrations (0.23-0.41 mm) were added to the perfusion solution.

Results: In the presence of 0.04 mm sevoflurane, the amplitude of population spikes was significantly depressed, and tetanic stimulation induced only posttetanic potentiation and then failure of LTP. These inhibitory effects were antagonized by bicuculline (10 [mu]m), a [gamma]-aminobutyric acid type A receptor antagonist. Sevoflurane at 0.23-0.41 mm further depressed the amplitude of field excitatory postsynaptic potentials in a dose-dependent manner and completely blocked LTP. Bicuculline only partially antagonized 0.41 mm sevoflurane-induced profound inhibition of LTP. Sevoflurane at 0.23-0.41 mm, but not at 0.04 mm, significantly increased paired pulse facilitation, suggesting that sevoflurane has presynaptic actions to reduce glutamate release from nerve terminals.     

Isoflurane Blocks Synaptic Plasticity in the Mouse Hippocampus

Background: The volatile anesthetic isoflurane depresses glutamatergic transmission. In this study, the authors investigated the effects of isoflurane on the induction of long-term potentiation (LTP) and long-term depression (LTD) in slices from the juvenile and adult mouse hippocampus. Both forms of synaptic plasticity involve the activation of glutamate receptors.

Methods: Field excitatory postsynaptic potentials and excitatory postsynaptic currents from neurons in the CA1 area were evoked by stimulation of the Schaffer collateral-commissural pathway. Two independent synaptic inputs were stimulated. Clinically relevant concentrations (0.2-0.3 mm) of isoflurane were added to the perfusion solution.

Results: Field excitatory postsynaptic potentials from slices of juvenile and adult mice were depressed to 37.3 +/- 6.1% and 58.3 +/- 7.4%, respectively, and excitatory postsynaptic currents were reduced to 36.7 +/- 5.4% by isoflurane. A brief tetanic stimulation (100 Hz, 1 s) induced stable LTP of field excitatory postsynaptic potentials. In the presence of isoflurane, tetanization failed to induce LTP. The effect of isoflurane on LTP induction was reversible and could be prevented by antagonizing [gamma]-aminobutyric acid type A receptors (GABAA). Low-frequency stimulation (1 Hz/900 pulses) induced LTD. In the presence of isoflurane, low-frequency stimulation failed to induce LTD.     

Short-Term Synaptic Plasticity in the Visual Cortex During Development

The maturation of short-term synaptic plasticity was studiedin slices of the visual cortex obtained from rats during thefIrst 47 days of postnatal life. Responses of cortical neuronsto repetitive stimulation of the white matter at frequencies>5 Hz were examined by recording intracellularly at the restingmembrane potential level. Paired-pulse facilitation, an increasein the excitatory intracellular response following an initialresponse, was present in     

Nitrous Oxide Impairs Electrophysiologic Recovery after Severe Hypoxia in Rat Hippocampal Slices

Background: Research has suggested that nitrous oxide may be harmful to ischemic neurons; however, the evidence for this is equivocal. The authors used rat hippocampal slices to examine the effects of nitrous oxide on neuronal hypoxic damage.

Methods: The evoked population spike (PS) was recorded from hippocampal CA1 pyramidal cells before, during, and after hypoxia. Control groups received nitrogen concentrations equal to nitrous oxide throughout the experiments. Biochemical measurements were made from dissected CA1 regions under experimental conditions that matched the electro-physiology studies.

Results: Recovery of the PS after hypoxia was 18 +/- 7% in slices treated with 50% nitrous oxide before and during 3.5 min of hypoxia; this compares with 41 +/- 9% (P < 0.05) in nitrogen-treated slices. Slices treated with nitrous oxide (95%) only during hypoxia (6 min) also demonstrated significantly less recovery of the PS than did slices treated with nitrogen. There was no significant difference in recovery if nitrous oxide was discontinued after the hypoxic period. Adenosine triphosphate concentrations after 3.5 min of hypoxia in slices treated with nitrous oxide decreased to the same extent as in nitrogen-treated slices (47% vs. 50%). Calcium influx increased during 10 min of hypoxia in untreated slices, but nitrous oxide did not significantly increase calcium influx during hypoxia. The sodium concentrations increased and potassium concentrations decreased during hypoxia; nitrous oxide did not significantly alter these changes.     

Effects of Propofol on Hippocampal Synaptic Transmission in Behaving Rats

Background: The action of propofol has been studied in vitro and in vivo, but the effects of intravenously administered propofol on synaptic transmission in freely behaving rats have not been studied before.

Methods: Rats were implanted with recording electrodes in the dentate gyrus and with stimulation electrodes in the medial perforant path (MPP). Paired pulses at different interpulse intervals (IPIs) were delivered to the MPP, and average evoked potentials were recorded in the dentate gyrus before and after a bolus of propofol (10 or 20 mg/kg administered intravenously) or control vehicle was injected via a femoral vein cannula. Because of the layered structure of the hippocampus, population excitatory postsynaptic potentials and population spikes could be distinguished and analyzed.

Results: Propofol has no significant effect on the population excitatory postsynaptic potentials or population spike evoked by a single MPP stimulus pulse. However, paired-pulse inhibition of the dentate population spikes was increased at IPI of 20 and 30 ms. Paired-pulse inhibition of the population spike was most prominent when tail pinch response was lost (deep and moderate anesthesia), but it persisted during light anesthesia. At 200 ms IPI, paired-pulse facilitation of population spikes was observed during moderate anesthesia in most rats.     

胰岛素抵抗综合征与麻醉

1胰岛素抵抗和胰岛素抵抗综合征 胰岛素抵抗（insulin resistance，IR）是胰岛素协助机体靶细胞（包括肝细胞、肌细胞、脂肪细胞和血管内皮细胞）摄取和利用葡萄糖的能力低下，即生理剂量的胰岛素产生低于正常生物学效应的一种状态。在早中期，机体为克服高血糖，往往代偿性分泌过多胰岛素，     

Antisense miR-7 Impairs Insulin Expression in Developing Pancreas and in Cultured Pancreatic Buds

MicroRNAs regulate gene expression by inhibiting translation or inducing target mRNA degradation. MicroRNAs regulate organ differentiation and embryonic development, including pancreatic specification and islet function. We showed previously that miR-7 is highly expressed in human pancreatic fetal and adult endocrine cells. Here we determined the expression profile of miR-7 in the mouse-developing pancreas by RT-PCR and in situ hybridization. MiR-7 expression was low between embryonic days e10.5 and e11.5, then began to increase at e13.5 through e14.5, and eventually decreased by e18. In situ hybridization and immunostaining analysis showed that miR-7 colocalizes with endocrine marker Isl1, suggesting that miR-7 is expressed preferentially in endocrine cells. Whole-mount in situ hybridization shows miR-7 highly expressed in the embryonic neural tube. To investigate the role of miR-7 in development of the mouse endocrine pancreas, antisense miR-7 morpholinos (MO) were delivered to the embryo at an early developmental stage (e10.5 days) via intrauterine fetal heart injection. Inhibition of miR-7 during early embryonic life results in an overall downregulation of insulin production, decreased β-cell numbers, and glucose intolerance in the postnatal period. This phenomenon is specific for miR-7 and possibly due to a systemic effect on pancreatic development. On the other hand, the in vitro inhibition of miR-7 in explanted pancreatic buds leads to β-cell death and generation of β-cells expressing less insulin than those in MO control. Therefore, in addition to the potential indirect effects on pancreatic differentiation derived from its systemic downregulation, the knockdown of miR-7 appears to have a β-cell-specific effect as well. These findings suggest that modulation of miR-7 expression could be utilized in the development of stem cell therapies to cure diabetes.     

小白菊内酯改善胰岛素抵抗的分子学机制

目的探讨小白菊内酯（PTL）对胰岛素抵抗（IR） 的作用及其分子学机制。方法用不同浓度的胰岛素对HepG2 细胞进行不同时间的诱导,通过葡萄糖氧化酶法测定HepG2 细胞葡萄糖含量,明确建立稳定的HepG2胰岛素抵抗模型的胰岛素诱导浓度及诱导时间。模型建立后,应用不同浓度PTL分别作用于IR细胞24 h,通过CCK8法评价细胞活性,葡萄糖氧化酶法测定IR模型HepG2细胞葡萄糖消耗量,明确PTL最佳作用浓度,Q PCR法检测细胞核因子κB（NF κB）及IκBα mRNA表达情况,免疫蛋白印迹技术检测蛋白IκBα表达情况。结果HepG2 细胞产生IR的最佳作用时间是24 h,最佳胰岛素浓度为10 μg·mL－1。PTL的最佳作用浓度为 20 μmol·L－1,应用其干预IR的HepG2细胞后,与IR的HepG2细胞相比,NF κB活性明显降低（P＜0.05）,IκBα降解被抑制（P＜0.05）。结论PTL可明显抑制IκBα降解,减少NF κB解离, 改善高胰岛素诱导产生的IR。     

Failure of Homeostatic Model Assessment of Insulin Resistance to Detect Marked Diet-Induced Insulin Resistance in Dogs

Accurate quantification of insulin resistance is essential for determining efficacy of treatments to reduce diabetes risk. Gold-standard methods to assess resistance are available (e.g., hyperinsulinemic clamp or minimal model), but surrogate indices based solely on fasting values have attractive simplicity. One such surrogate, the homeostatic model assessment of insulin resistance (HOMA-IR), is widely applied despite known inaccuracies in characterizing resistance across groups. Of greater significance is whether HOMA-IR can detect changes in insulin sensitivity induced by an intervention. We tested the ability of HOMA-IR to detect high-fat diet–induced insulin resistance in 36 healthy canines using clamp and minimal model analysis of the intravenous glucose tolerance test (IVGTT) to document progression of resistance. The influence of pancreatic function on HOMA-IR accuracy was assessed using the acute insulin response during the IVGTT (AIR_G). Diet-induced resistance was confirmed by both clamp and minimal model (P < 0.0001), and measures were correlated with each other (P = 0.001). In striking contrast, HOMA-IR ([fasting insulin (μU/mL) × fasting glucose (mmol)]/22.5) did not detect reduced sensitivity induced by fat feeding (P = 0.22). In fact, 13 of 36 animals showed an artifactual decrease in HOMA-IR (i.e., increased sensitivity). The ability of HOMA-IR to detect diet-induced resistance was particularly limited under conditions when insulin secretory function (AIR_G) is less than robust. In conclusion, HOMA-IR is of limited utility for detecting diet-induced deterioration of insulin sensitivity quantified by glucose clamp or minimal model. Caution should be exercised when using HOMA-IR to detect insulin resistance when pancreatic function is compromised. It is necessary to use other accurate indices to detect longitudinal changes in insulin resistance with any confidence.     

Metabolic Syndrome and Insulin Resistance: Perioperative Considerations

Metabolic syndrome represents a constellation of risk factors associated with increased incidence of cardiovascular disease and progression to diabetes mellitus. Insulin resistance, a state of decreased biologic response to physiologic concentrations of insulin, is a key component of this syndrome and seems to be the result of a primary defect at the skeletal muscle glucose transporter. Acute illness and the perioperative period are characterized by a state of insulin resistance that manifests as hyperglycemia and leads to various other metabolic and biochemical alterations that adversely affect end organ function. Hyperglycemia in acutely ill patients adversely affects outcome. Achieving euglycemia seems beneficial in certain clinical situations, but considerable disagreement exists regarding the target blood sugar levels, the duration of therapy, and the modality. Pharmacotherapy, exercise, and nutrition to improve insulin sensitivity seem promising but require further evaluation to confirm their efficacy for perioperative risk reduction. This review discusses the pathophysiology and the clinical implications of metabolic syndrome and insulin resistance in the acutely ill patient with an emphasis on perioperative modulation strategies.