Intermittent Fasting Reduces Neuroinflammation and Cognitive Impairment in High-Fat Diet-Fed Mice by Downregulating Lipocalin-2 and Galectin-3.

Intermittent fasting (IF), an alternating pattern of dietary restriction, reduces obesity-induced insulin resistance and inflammation. However, the crosstalk between adipose tissue and the hippocampus in diabetic encephalopathy is not fully understood. Here, we investigated the protective effects of IF against neuroinflammation and cognitive impairment in high-fat diet(HFD)-fed mice. Histological analysis revealed that IF reduced crown-like structures and adipocyte apoptosis in the adipose tissue of HFD mice. In addition to circulating lipocalin-2 (LCN2) and galectin-3 (GAL3) levels, IF reduced HFD-induced increases in LCN2- and GAL3-positive macrophages in adipose tissue. IF also improved HFD-induced memory deficits by inhibiting blood-brain barrier breakdown and neuroinflammation. Furthermore, immunofluorescence showed that IF reduced HFD-induced astrocytic LCN2 and microglial GAL3 protein expression in the hippocampus of HFD mice. These findings indicate that HFD-induced adipocyte apoptosis and macrophage infiltration may play a critical role in glial activation and that IF reduces neuroinflammation and cognitive impairment by protecting against blood-brain barrier leakage.

Intermittent Fasting Attenuates Metabolic-Dysfunction-Associated Steatohepatitis by Enhancing the Hepatic Autophagy-Lysosome Pathway.

An intermittent fasting (IF) regimen has been shown to protect against metabolic dysfunction-associated steatohepatitis (MASH). However, the precise mechanism remains unclear. Here, we explored how IF reduced hepatic lipid accumulation, inflammation, and fibrosis in mice with MASH. The mice were fed a high-fat diet (HFD) for 30 weeks and either continued on the HFD or were subjected to IF for the final 22 weeks. IF reduced body weight, insulin resistance, and hepatic lipid accumulation in HFD-fed mice. Lipidome analysis revealed that IF modified HFD-induced hepatic lipid composition. In particular, HFD-induced impaired autophagic flux was reversed by IF. The decreased hepatic lysosome-associated membrane protein 1 level in HFD-fed mice was upregulated in HFD+IF-fed mice. However, increased hepatic lysosomal acid lipase protein levels in HFD-fed mice were reduced by IF. IF attenuated HFD-induced hepatic inflammation and galectin-3-positive Kupffer cells. In addition to the increases in hepatic hydroxyproline and lumican levels, lipocalin-2-mediated signaling was reversed in HFD-fed mice by IF. Taken together, our findings indicate that the enhancement of the autophagy-lysosomal pathway may be a critical mechanism of MASH reduction by IF.

TonEBP Haploinsufficiency Attenuates Microglial Activation and Memory Deficits in Middle-Aged and Amyloid ß Oligomer-Treated Mice.

Age-related microglial activation is associated with cognitive impairment. Tonicity-responsive enhancer-binding protein (TonEBP) is a critical mediator of microglial activation in response to neuroinflammation. However, the precise role of TonEBP in the middle-aged brain is not yet known. We used TonEBP haploinsufficient mice to investigate the role of TonEBP in middle-aged or amyloid ß oligomer (AßO)-injected brains and examined the effect of TonEBP knockdown on AßO-treated BV2 microglial cells. Consistent with an increase in microglial activation with aging, hippocampal TonEBP expression levels were increased in middle-aged (12-month-old) and old (24-month-old) mice compared with young (6-month-old) mice. Middle-aged TonEBP haploinsufficient mice showed reduced microglial activation and fewer memory deficits than wild-type mice. Electron microscopy revealed that synaptic pruning by microglial processes was reduced by TonEBP haploinsufficiency. TonEBP haploinsufficiency also reduced dendritic spine loss and improved memory deficits in AßO-treated mice. Furthermore, TonEBP knockdown attenuated migration and phagocytosis in AßO-treated BV2 cells. These findings suggest that TonEBP plays important roles in age-related microglial activation and memory deficits.

Amyloid ß oligomer promotes microglial galectin-3 and astrocytic lipocalin-2 levels in the hippocampus of mice fed a high-fat diet.

Type 2 diabetes is associated with a risk factor for Alzheimer's disease (AD). Activation of glial cells, such as microglia and astrocytes, is crucial for the development of neuroinflammation in both diabetes and AD. The role of amyloid-beta oligomer (AßO) in the hippocampus of diabetic mice has been investigated; however, the effect of galectin-3 and lipocalin-2 (LCN2) on amyloid toxicity-related glial activation in diabetic mice is not known. To fill this knowledge gap, we fed mice a high-fat diet (HFD) for 20 weeks to induce a diabetic state and then injected the hippocampus with AßO. Sholl analysis of iba-1-positive microglia showed retraction of microglial ramifications in the hippocampus of HFD-fed diabetic mice. AßO treatment caused more retraction of microglial process in HFD-fed mice. In particular, microglial galectin-3 levels and astrocytic LCN2 levels were increased in the hippocampus of HFD-fed mice with AßO treatment. These findings suggest that galectin-3 and LCN2 are involved in amyloid toxicity mechanisms, especially glial activation under diabetic conditions.

Lipocalin-2 promotes acute lung inflammation and oxidative stress by enhancing macrophage iron accumulation.

Lipocalin-2 (LCN2) is an acute-phase protein that regulates inflammatory responses to bacteria or lipopolysaccharide (LPS). Although the bacteriostatic role of LCN2 is well studied, the function of LCN2 in acute lung damage remains unclear. Here, LCN2 knockout (KO) mice were used to investigate the role of LCN2 in LPS-treated mice with or without recombinant LCN2 (rLCN2). In addition, we employed patients with pneumonia. RAW264.7 cells were given LCN2 inhibition or rLCN2 with or without iron chelator deferiprone. LCN2 KO mice had a higher survival rate than wild-type (WT) mice after LPS treatment. In addition to elevated LCN2 levels in serum and bronchoalveolar lavage fluid (BALF), LPS treatment also increased LCN2 protein in alveolar macrophage lysates of BALF. LCN2 deletion attenuated neutrophil and macrophage infiltration in the lungs of LPS-treated mice as well as serum and BALF interleukin-6 (IL-6). Circulating proinflammatory cytokines and LCN2-positive macrophages were prominently increased in the BALF of pneumonia patients. In addition to increase of iron-stained macrophages in pneumonia patients, increased iron-stained macrophages and oxidative stress in LPS-treated mice were inhibited by LCN2 deletion. In contrast, rLCN2 pretreatment aggravated lung inflammation and oxidative stress in LPS-treated WT mice and then resulted in higher mortality. In RAW264.7 cells, exogenous LCN2 treatment also increased inflammation and oxidative stress, whereas LCN2 knockdown markedly diminished these effects. Furthermore, deferiprone inhibited inflammation, oxidative stress, and phagocytosis in RAW264.7 cells with high LCN2 levels, as well as LPS-induced acute lung injury in WT and LCN2 KO mice. Thus, these findings suggest that LCN2 plays a key role in inflammation and oxidative stress following acute lung injury and that LCN2 is a potential therapeutic target for pneumonia or acute lung injury.

Metformin-induced TTP mediates communication between Kupffer cells and hepatocytes to alleviate hepatic steatosis by regulating lipophagy and necroptosis.

OBJECTIVE: Emerging evidence suggests that crosstalk between Kupffer cells (KCs) and hepatocytes protects against non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms that lead to the reduction of steatosis in NAFLD remain obscure. METHODS: Ttp+/+ and Ttp-/- mice were fed with a high-fat diet. Hepatic steatosis was analyzed by Nile Red staining and measurement of inflammatory cytokines. Lipid accumulation and cell death were evaluated in co-culture systems with primary hepatocytes and KCs derived from either Ttp+/+ or Ttp-/- mice. RESULTS: Tristetraprolin (TTP), an mRNA binding protein, was essential for the protective effects of metformin in NAFLD. Metformin activated TTP via the AMPK-Sirt1 pathway in hepatocytes and KCs. TTP inhibited TNF-α production in KCs, which in turn decreased hepatocyte necroptosis. Downregulation of Rheb expression by TTP promoted hepatocyte lipophagy via mTORC1 inhibition and increased nuclear translocation of transcription factor-EB (TFEB). Consistently, TTP-deficient NAFLD mice failed to respond to metformin with respect to alleviation of hepatic steatosis, protection of hepatocyte necroptosis, or induction of lipophagy. CONCLUSIONS: TTP, which is essential for the protective effects of metformin, may represent a novel primary therapeutic target in NAFLD.

Lipocalin-2 deletion attenuates lipopolysaccharide-induced acute lung inflammation via downregulating chemotaxis-related genes.

Lipocalin-2 (LCN2) is an acute phase protein used as a biomarker for acute lung injury (ALI). Although the innate immune functions of LCN2 have been studied, how LCN2 contributes to ALI induced by lipopolysaccharide (LPS) remains unknown. In this study, we investigated the effect of LCN2 deletion on LPS-induced ALI using RNA-sequencing. LPS-treated LCN2 knockout (KO) mice had a decreased histopathological score and reduced neutrophil and macrophage infiltration in lung tissue compared with LPS-treated WT mice. RNA-sequencing analysis identified 38 differentially expressed genes (DEGs), including Cxcl5, Cxcl13, Xcl1, Saa1, and Cd14. In particular, Gene Ontology analysis of DEGs revealed a significant reduction in the inflammatory response, neutrophil chemotaxis, and chemokine-mediated signaling in LPS-treated LCN2KO mice compared with LPS-treated WT mice. Thus, these results suggest that LCN2 deletion alleviates LPS-induced ALI and that LCN2 may be involved in chemotaxis-related gene expression.

Lipocalin-2 activates hepatic stellate cells and promotes nonalcoholic steatohepatitis in high-fat diet-fed Ob/Ob mice.

BACKGROUND AND AIMS: In obesity and type 2 diabetes mellitus, leptin promotes insulin resistance and contributes to the progression of NASH via activation of hepatic stellate cells (HSCs). However, the pathogenic mechanisms that trigger HSC activation in leptin-deficient obesity are still unknown. This study aimed to determine how HSC-targeting lipocalin-2 (LCN2) mediates the transition from simple steatosis to NASH. APPROACH AND RESULTS: Male wild-type (WT) and ob/ob mice were fed a high-fat diet (HFD) for 20 weeks to establish an animal model of NASH with fibrosis. Ob/ob mice were subject to caloric restriction or recombinant leptin treatment. Double knockout (DKO) mice lacking both leptin and lcn2 were also fed an HFD for 20 weeks. In addition, HFD-fed ob/ob mice were treated with gadolinium trichloride to deplete Kupffer cells. The LX-2 human HSCs and primary HSCs from ob/ob mice were used to investigate the effects of LCN2 on HSC activation. Serum and hepatic LCN2 expression levels were prominently increased in HFD-fed ob/ob mice compared with normal diet-fed ob/ob mice or HFD-fed WT mice, and these changes were closely linked to liver fibrosis and increased hepatic α-SMA/matrix metalloproteinase 9 (MMP9)/signal transducer and activator of transcription 3 (STAT3) protein levels. HFD-fed DKO mice showed a marked reduction of α-SMA protein compared with HFD-fed ob/ob mice. In particular, the colocalization of LCN2 and α-SMA was increased in HSCs from HFD-fed ob/ob mice. In primary HSCs from ob/ob mice, exogenous LCN2 treatment induced HSC activation and MMP9 secretion. By contrast, LCN2 receptor 24p3R deficiency or a STAT3 inhibitor reduced the activation and migration of primary HSCs. CONCLUSIONS: LCN2 acts as a key mediator of HSC activation in leptin-deficient obesity via α-SMA/MMP9/STAT3 signaling, thereby exacerbating NASH.

Adzuki Bean MY59 Extract Reduces Insulin Resistance and Hepatic Steatosis in High-Fat-Fed Mice via the Downregulation of Lipocalin-2.

Adzuki bean is well known as a potential functional food that improves metabolic complications from obesity and diabetes. Lipocalin-2 (LCN2) has been implicated to have an important role in obesity and diabetes. However, the protective roles of adzuki bean MY59 extract (ABE) on insulin resistance and hepatic steatosis are not fully understood. In the present study, we investigated the effects of ABE on LCN2 expression in high-fat diet (HFD)-fed mice. ABE reduced HFD-induced fat mass and improved insulin resistance. In addition to hepatic steatosis, HFD-fed mice showed many apoptotic cells and neutrophils in the epididymal fat pads. However, these findings were significantly reduced by ABE supplementation. In particular, we found that increased LCN2 proteins from serum, epididymal fat pads, and liver in HFD-fed mice are significantly reduced by ABE. Furthermore, ABE reduced increased heme oxygenase-1 and superoxide dismutase-1 expressions in adipose tissue and liver in HFD-fed mice. We found that hepatic nuclear factor-kappa B (NF-κB) p65 expression in HFD-fed mice was also reduced by ABE. Thus, these findings indicate that ABE feeding could improve insulin resistance and hepatic steatosis by decreasing LCN2-mediated inflammation and oxidative stress in HFD-fed mice.

Lipocalin-2 Deficiency Reduces Hepatic and Hippocampal Triggering Receptor Expressed on Myeloid Cells-2 Expressions in High-Fat Diet/Streptozotocin-Induced Diabetic Mice.

BACKGROUND: Lipocalin-2 (LCN2) is an acute-phase protein that has been linked to insulin resistance, diabetes, and neuroinflammatory diseases. Triggering receptor expressed on myeloid cells-2 (TREM2) has been also implicated in microglia-mediated neuroinflammation. However, the potential role of LCN2 on TREM2 in diabetic mouse models is not fully understood. METHODS: We investigated hepatic and hippocampal TREM2 expressions in high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic LCN2 knockout (KO) mice. RESULTS: In addition to increased serum LCN2 level, diabetic wild-type (WT) mice had insulin resistance and hepatic steatosis. However, LCN2 deletion attenuated these metabolic parameters in diabetic mice. We also found that LCN2 deletion reduced hepatic inflammation and microglial activation in diabetic mice. In particular, diabetic LCN2 KO mice had a reduction in hepatic and hippocampal TREM2 expressions compared with diabetic WT mice. Furthermore, we found that many TREM2-positive Kupffer cells and microglia in diabetic WT mice were reduced through LCN2 deletion. CONCLUSIONS: These findings indicate that LCN2 may promote hepatic inflammation and microglial activation via upregulation of TREM2 in diabetic mice.

LCN2 deficiency ameliorates doxorubicin-induced cardiomyopathy in mice.

Doxorubicin (DOX) is an effective anticancer drug with the side effect of irreparable cardiomyopathy. Lipocalin-2 (LCN2) has been identified as an important regulator of oxidative stress and inflammation in cardiovascular disease pathophysiology. Here, we demonstrate that LCN2 deletion increases autophagic flux in the DOX-treated hearts. Mice were injected intraperitoneally six times with 30 mg/kg DOX. Echocardiography showed that DOX-treated wild-type (WT) mice had markedly weaker cardiac function compared to saline-treated WT mice. In DOX-treated LCN2 knockout (KO) mice, cardiac function was partially restored. Histological analysis showed a reduction in cardiomyocyte diameter in DOX-treated WT mice that was ameliorated in DOX-treated LCN2KO mice. Cardiac levels of phosphorylated signal transducer and activator of transcription 3, LCN2, heme oxygenase-1, and NAD (P) H dehydrogenase were markedly greater in DOX-treated WT mice than in DOX-treated LCN2KO mice. Light chain 3B (LC3B)II expression was higher in DOX-treated WT mice, but lower in DOX-treated LCN2KO mice when compared to saline-treated WT mice. Less co-localization of LC3B and lysosomal-associated membrane protein 1 was observed in DOX-treated WT mice than in DOX-treated LCN2KO mice. LCN2 co-localized with LC3B-stained cells in the DOX-treated WT mouse heart, but not in the DOX-treated LCN2KO mouse heart. These findings indicate that the cardiotoxic effect of DOX is due to autophagosome accumulation mediated by LCN2 upregulation and that LCN2 may inhibit autophagic flux, leading to DOX-induced cardiomyopathy.

Role of Lipocalin-2 in Amyloid-Beta Oligomer-Induced Mouse Model of Alzheimer's Disease.

Lipocalin-2 (LCN2) is an inflammatory protein with diverse functions in the brain. Although many studies have investigated the mechanism of LCN2 in brain injuries, the effect of LCN2 on amyloid-toxicity-related memory deficits in a mouse model of Alzheimer's disease (AD) has been less studied. We investigated the role of LCN2 in human AD patients using a mouse model of AD. We created an AD mouse model by injecting amyloid-beta oligomer (AßO) into the hippocampus. In this model, animals exhibited impaired learning and memory. We found LCN2 upregulation in the human brain frontal lobe, as well as a positive correlation between white matter ischemic changes and serum LCN2. We also found increased astrocytic LCN2, microglia activation, iron accumulation, and blood-brain barrier disruption in AßO-treated hippocampi. These findings suggest that LCN2 is involved in a variety of amyloid toxicity mechanisms, especially neuroinflammation and oxidative stress.

Tonicity-responsive enhancer-binding protein promotes diabetic neuroinflammation and cognitive impairment via upregulation of lipocalin-2.

BACKGROUND: Diabetic individuals have increased circulating inflammatory mediators which are implicated as underlying causes of neuroinflammation and memory deficits. Tonicity-responsive enhancer-binding protein (TonEBP) promotes diabetic neuroinflammation. However, the precise role of TonEBP in the diabetic brain is not fully understood. METHODS: We employed a high-fat diet (HFD)-only fed mice or HFD/streptozotocin (STZ)-treated mice in our diabetic mouse models. Circulating TonEBP and lipocalin-2 (LCN2) levels were measured in type 2 diabetic subjects. TonEBP haploinsufficient mice were used to investigate the role of TonEBP in HFD/STZ-induced diabetic mice. In addition, RAW 264.7 macrophages were given a lipopolysaccharide (LPS)/high glucose (HG) treatment. Using a siRNA, we examined the effects of TonEBP knockdown on RAW264 cell' medium/HG-treated mouse hippocampal HT22 cells. RESULTS: Circulating TonEBP and LCN2 levels were higher in experimental diabetic mice or type 2 diabetic patients with cognitive impairment. TonEBP haploinsufficiency ameliorated the diabetic phenotypes including adipose tissue macrophage infiltrations, neuroinflammation, blood-brain barrier leakage, and memory deficits. Systemic and hippocampal LCN2 proteins were reduced in diabetic mice by TonEBP haploinsufficiency. TonEBP (+ / -) mice had a reduction of hippocampal heme oxygenase-1 (HO-1) expression compared to diabetic wild-type mice. In particular, we found that TonEBP bound to the LCN2 promoter in the diabetic hippocampus, and this binding was abolished by TonEBP haploinsufficiency. Furthermore, TonEBP knockdown attenuated LCN2 expression in lipopolysaccharide/high glucose-treated mouse hippocampal HT22 cells. CONCLUSIONS: These findings indicate that TonEBP may promote neuroinflammation and cognitive impairment via upregulation of LCN2 in diabetic mice.

Exendin-4 Pretreatment Attenuates Kainic Acid-Induced Hippocampal Neuronal Death.

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that protects against brain injury. However, little is known about the effect of Ex-4 on kainic acid (KA)-induced seizures and hippocampal cell death. Therefore, this study evaluated the neuroprotective effects of Ex-4 pretreatment in a mouse model of KA-induced seizures. Three days before KA treatment, mice were intraperitoneally injected with Ex-4. We found that Ex-4 pretreatment reversed KA-induced reduction of GLP-1R expression in the hippocampus and attenuated KA-induced seizure score, hippocampal neuronal death, and neuroinflammation. Ex-4 pretreatment also dramatically reduced hippocampal lipocalin-2 protein in KA-treated mice. Furthermore, immunohistochemical studies showed that Ex-4 pretreatment significantly alleviated blood-brain barrier leakage. Finally, Ex-4 pretreatment stimulated hippocampal expression of phosphorylated cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), a known target of GLP-1/GLP-1R signaling. These findings indicate that Ex-4 pretreatment may protect against KA-induced neuronal damage by regulating GLP-1R/CREB-mediated signaling pathways.

Skeletal Lipocalin-2 Is Associated with Iron-Related Oxidative Stress in ob/ob Mice with Sarcopenia.

Obesity and insulin resistance accelerate aging-related sarcopenia, which is associated with iron load and oxidative stress. Lipocalin-2 (LCN2) is an iron-binding protein that has been associated with skeletal muscle regeneration, but details regarding its role in obese sarcopenia remain unclear. Here, we report that elevated LCN2 levels in skeletal muscle are linked to muscle atrophy-related inflammation and oxidative stress in leptin-deficient ob/ob mice. RNA sequencing analyses indicated the LCN2 gene expression is enhanced in skeletal muscle of ob/ob mice with sarcopenia. In addition to muscular iron accumulation in ob/ob mice, expressions of iron homeostasis-related divalent metal transporter 1, ferritin, and hepcidin proteins were increased in ob/ob mice compared to lean littermates, whereas expressions of transferrin receptor and ferroportin were reduced. Collectively, these findings demonstrate that LCN2 functions as a potent proinflammatory factor in skeletal muscle in response to obesity-related sarcopenia and is thus a therapeutic candidate target for sarcopenia treatment.

Ablation of dynamin-related protein 1 promotes diabetes-induced synaptic injury in the hippocampus.

Dynamin-related protein 1 (Drp1)-mediated mitochondrial dysfunction is associated with synaptic injury in the diabetic brain. However, the dysfunctional mitochondria by Drp1 deletion in the diabetic brain are poorly understood. Here, we investigated the effects of neuron-specific Drp1 deletion on synaptic damage and mitophagy in the hippocampus of a high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. HFD/STZ-induced diabetic mice exhibited metabolic disturbances and synaptic damages. Floxed Drp1 mice were crossed with Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα)-Cre mice, to generate neuron-specific Drp1 knockout (Drp1cKO) mice, which showed marked mitochondrial swelling and dendritic spine loss in hippocampal neurons. In particular, diabetic Drp1cKO mice exhibited an increase in dendritic spine loss and higher levels of oxidative stress and neuroinflammation compared with diabetic wild-type (WT) mice. Diabetic WT mice generally displayed increased Drp1-induced small mitochondrial morphology in hippocampal neurons, but large mitochondria were prominently observed in diabetic Drp1cKO mice. The levels of microtubule-associated protein 1 light-chain 3 and lysosomal-associated membrane protein 1 proteins were significantly increased in the hippocampus of diabetic Drp1cKO mice compared with diabetic WT mice. The inhibition of Drp1 adversely promotes synaptic injury and neurodegeneration in the diabetic brain. The findings suggest that the exploratory mechanisms behind Drp1-mediated mitochondrial dysfunction could provide a possible therapeutic target for diabetic brain complications.

Lipocalin-2 Deficiency Reduces Oxidative Stress and Neuroinflammation and Results in Attenuation of Kainic Acid-Induced Hippocampal Cell Death.

The hippocampal cell death that follows kainic acid (KA)-induced seizures is associated with blood-brain barrier (BBB) leakage and oxidative stress. Lipocalin-2 (LCN2) is an iron-trafficking protein which contributes to both oxidative stress and inflammation. However, LCN2's role in KA-induced hippocampal cell death is not clear. Here, we examine the effect of blocking LCN2 genetically on neuroinflammation and oxidative stress in KA-induced neuronal death. LCN2 deficiency reduced neuronal cell death and BBB leakage in the KA-treated hippocampus. In addition to LCN2 upregulation in the KA-treated hippocampus, circulating LCN2 levels were significantly increased in KA-treated wild-type (WT) mice. In LCN2 knockout mice, we found that the expressions of neutrophil markers myeloperoxidase and neutrophil elastase were decreased compared to their expressions in WT mice following KA treatment. Furthermore, LCN2 deficiency also attenuated KA-induced iron overload and oxidative stress in the hippocampus. These findings indicate that LCN2 may play an important role in iron-related oxidative stress and neuroinflammation in KA-induced hippocampal cell death.

The Role of SHIP1 on Apoptosis and Autophagy in the Adipose Tissue of Obese Mice.

Obesity-induced adipocyte apoptosis promotes inflammation and insulin resistance. Src homology domain-containing inositol 5'-phosphatase 1 (SHIP1) is a key factor of apoptosis and inflammation. However, the role of SHIP1 in obesity-induced adipocyte apoptosis and autophagy is unclear. We found that diet-induced obesity (DIO) mice have significantly greater crown-like structures and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL)-positive cells than ob/ob or control mice. Using RNA sequencing (RNA-seq) analysis, we identified that the apoptosis- and inflammation-related gene Ship1 is upregulated in DIO and ob/ob mice compared with control mice. In particular, DIO mice had more SHIP1-positive macrophages and lysosomal-associated membrane protein 1 (LAMP1) as well as a higher B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio compared with ob/ob or control mice. Furthermore, caloric restriction attenuated adipose tissue inflammation, apoptosis, and autophagy by reversing increases in SHIP1-associated macrophages, Bax/Bcl2-ratio, and autophagy in DIO and ob/ob mice. These results demonstrate that DIO, not ob/ob, aggravates adipocyte inflammation, apoptosis, and autophagy due to differential SHIP1 expression. The evidence of decreased SHIP1-mediated inflammation, apoptosis, and autophagy indicates new therapeutic approaches for obesity-induced chronic inflammatory diseases.

Hippocampal Lipocalin 2 Is Associated With Neuroinflammation and Iron-Related Oxidative Stress in ob/ob Mice.

Obesity causes brain injuries with inflammatory and structural changes, leading to neurodegeneration. Although increased circulating lipocalin 2 (LCN2) level has been implicated in neurodegenerative diseases, the precise mechanism of neurodegeneration in obesity is not clear. Here, we investigated whether LCN2-mediated signaling promotes neurodegeneration in the hippocampus of leptin-deficient ob/ob mice, which are characterized by obesity, insulin resistance, systemic inflammation, and neuroinflammation. In particular, there was significant upregulation of both LCN2 and matrix metalloproteinase 9 levels from serum and hippocampus in ob/ob mice. Using RNA-seq analysis, we found that neurodegeneration- sortilin-related receptor 1 (Sorl1) and brain-derived neurotrophic factor (Bdnf) genes were significantly reduced in the hippocampus of ob/ob mice. We additionally found that the endosome-related WD repeat and FYVE-domain-containing 1 (Wdfy1) gene were upregulated in ob/ob mice. In particular, iron overload-related mitochondrial ferritin and nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) proteins were increased in the hippocampus of ob/ob. Thus, these findings indicate that iron-binding protein LCN2-mediated oxidative stress promotes neurodegeneration in ob/ob mice.