HFD-fed KO Mice Research Articles

LRRK2 negatively regulates glucose tolerance via regulation of membrane translocation of GLUT4 in adipocytes.

Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high-fat diet (HFD)-induced glucose intolerance using Lrrk2-knockout (KO) mice. We found for the first time that HFD-fed KO mice display improved glucose tolerance compared with their wild-type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin-related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin-dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3-L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8- and Rab10-mediated GLUT4 membrane translocation.

Gpr75-deficient mice are protected from high-fatdiet-induced obesity.

G-protein coupled receptor 75 (GPR75) has been identified as the high-affinity receptor of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive and proinflammatory lipid, and mice overproducing 20-HETE have been shown to develop insulin resistance when fed a high-fat diet (HFD), which was prevented by a 20-HETE receptor blocker. Simultaneously, a large-scale exome sequencing of 640,000 subjects identified an association between loss-of-function GPR75 variants and protection against obesity. Wild-type (WT) and Gpr75-deficient mice were placed on HFD for 14 weeks, and their obesity phenotype was examined. Male and female Gpr75 null (knockout [KO]) and heterozygous mice gained less weight than WT mice when placed on HFD. KO mice maintained the same level of energy expenditure during HFD feeding, whereas WT mice showed a significant reduction in energy expenditure. Diet-driven adiposity and adipocyte hypertrophy were greatly lessened in Gpr75-deficient mice. HFD-fed KO mice did not develop insulin resistance. Adipose tissue from Gpr75-deficient mice had increased expression of thermogenic genes and decreased levels of inflammatory markers. Moreover, insulin signaling, which was impaired in HFD-fed WT mice, was unchanged in KO mice. These findings suggest that GPR75 is an important player in the control of metabolism and glucose homeostasis and a likely novel therapeutic target to combat obesity-driven metabolic disorders.

Macrophage LAMTOR1 Deficiency Prevents Dietary Obesity and Insulin Resistance Through Inflammation-Induced Energy Expenditure.

Here, we studied the metabolic function of LAMTOR1 from macrophages using LAMTOR1 macrophage-specific knockout (MKO) mice. LAMTOR1 MKO mice showed resistance to high-fat diet (HFD)-induced obesity, lipid steatosis, and glucose metabolic disorders, with elevated levels of pro-inflammatory cytokines. The energy expenditure, oxygen consumption, and CO2 production increased significantly in HFD-fed MKO vs. wild-type (WT) mice. HE and immunohistochemistry staining showed a remarkable CD68+ Kupffer cell accumulation in the liver. Additionally, flow cytometry revealed that the proportion of macrophages and monocytes increased significantly in the liver of MKO mice. Of note, these macrophages were probably derived from the bone marrow since the proportion of CD11b+ cells as well as the proliferative activity was also increased in the context of femoral bone marrow cells. In addition, the Kupffer cells of both WT and KO mice were double-positive for the M1 (CD86) and M2 (CD206) markers. However, the expression of both M1 and M2 macrophage-related genes was increased in the liver of HFD-fed KO mice. Murine primary hepatocytes and Kupffer cells were further isolated and incubated with oleic acid for 24 h. The glucose output of primary hepatocytes from MKO mice was not affected. However, decreased lipid tolerance was observed in LAMTOR1-deficient Kupffer cells. Overall, our results suggest that LAMTOR1 deficiency in macrophages prevents obesity and metabolic disorders via the accumulation of Kupffer cells in the liver and the consequent hyper-inflammation and increased energy expenditure. Therefore, our results provide a new perspective for macrophage-derived LAMTOR1 in the context of systemic metabolism.

265-LB: Mitochondria-Targeted DNA Repair Glycosylase OGG1 Protects Against HFD-Induced Obesity and Insulin Resistance in OGG1 Deficient Mice

Mitochondrial DNA (mtDNA) damage heightens mitochondrial oxidative stress, which is very critical for the development of insulin resistance. OGG1 (8-oxoguanine DNA glycosylase-1) is a DNA glycosylase mediating the first step in the base excision repair which removes 7,8-dihydro-8-oxoguanine (8-oxoG) and repairs oxidized nuclear and mtDNA. Previous studies showed that Ogg1 deficiency results in an increased susceptibility to high fat diet (HFD)-induced obesity, metabolic dysfunction and IR in mice, suggesting a crucial role of Ogg1 in glucose metabolism. Here we performed studies in vivo using Ogg1 knockout and overexpressing genetic models: 1)Ogg1 -/- (KO); 2)KO mice with mitochondrial targeted hOGG1 (hOGG1, subunit 1-α; KO/Tg); 3) wild type (WT) mice. Male mice of all three genotypes were fed either high fat diet (HFD) or control low fat diet (LFD) for 16 weeks, and obesity and insulin resistance phenotypes were evaluated. Our results demonstrated that HFD-fed KO mice displayed increased obesity and whole-body insulin resistance phenotype compared to both WT and KO/Tg mice fed with HFD. Additionally, we have utilized a translational strategy of targeting the recombinant hOGG1 into mitochondria using a TAT-protein transduction system. KO mice were fed HFD and vehicle or MTS-hOGG1-TAT protein was administered intraperitoneally twice weekly for 9 weeks. Consistent with results obtained for genetic models, KO mice injected with MTS-hOGG1-TAT showed significantly lower blood glucose levels and insulin resistance phenotype compared to mice injected with vehicle. Our findings suggest that protecting mtDNA from damage might be crucial to prevent insulin resistance. Furthermore, our data provides strong rationale to use TAT-delivery of DNA repair enzymes into mitochondria as a new translational approach for treatment of insulin resistance. Disclosure L. Yuzefovych: None. L. Rachek: None. Funding American Heart Association (16GRNT29150012 to L.R.)

DNA Repair Enzyme Ogg1 Regulates Hepatic Insulin Resistance in High-Fat Diet-Fed Obese Mice

Mitochondrial DNA (mtDNA) damage has been implicated in the development of insulin resistance. The mtDNA is highly specialized and encodes for proteins essential for energy metabolism. Also, mtDNA damage heightens mitochondrial oxidative stress, which is very critical for insulin resistance. OGG1 (8-oxoguanine DNA glycosylase-1) is a DNA glycosylase mediating the first step in the base excision repair which removes 7,8-dihydro-8-oxoguanine (8-oxoG) and repairs oxidized nuclear and mitochondrial DNA. Previous studies showed that Ogg1 deficiency results in an increased susceptibility to high fat diet (HFD)-induced obesity, metabolic dysfunction and insulin resistance in mice, suggesting a crucial role of Ogg1 in glucose metabolism. In the current study, we performed a 2-hour hyperinsulinemic-euglycemic clamp to measure tissue-specific insulin sensitivity in wild type (WT) and Ogg1-/- (KO) mice after chronic feeding of low fat diet (LFD as controls) or HFD. On LFD, both WT and KO mice showed comparable body weight and insulin sensitivity. After 16 weeks of HFD, the KO mice were more obese than WT mice with significant increases in whole body fat mass. There was a strong trend of increased insulin resistance with lower glucose infusion rates and whole body glucose turnover and glycogen synthesis in HFD-fed KO mice compared to HFD-fed WT mice. Hepatic insulin action was significantly lower in the HFD-fed KO mice which was consistent with recent evidence showing Ogg1 regulation of hepatic gluconeogenesis in the fed state. This is the first evidence demonstrating that Ogg1 contributes to HFD-induced insulin resistance in liver. Our findings suggest that protecting mtDNA from damage might be crucial to prevent insulin resistance and further identify therapeutic strategies for stimulating OGG1 as potential treatment of insulin resistance. Disclosure L. Yuzefovych: None. M. Schuler: None. H. Noh: None. S. Suk: None. J.K. Kim: None. L. Rachek: None.

Abstract P347: Retinol-binding Protein 7 (RBP7) is Required for PPARG-mediated Endothelial Protection via Adiponectin

PPARγ protects against endothelial dysfunction by regulation of unknown target genes. One such target, RBP7, an intracellular fatty acid-binding protein, exhibits endothelium-specific expression, but its effect on vascular function remain unknown. We hypothesize that RBP7 is endothelial protective. We examined vascular responses in basilar artery (pressurized myograph) of RBP7-knockout (KO) and wild type (WT) mice fed normal chow (ND) or high fat diet (HFD) for 8 wks. Endothelium-dependent acetylcholine (ACh)-induced relaxation was significantly impaired in HFD-fed KO mice (ACh, 100μM: 33±7% KO vs 83±10% WT, p<0.05), but not in ND-fed groups. This response was ameliorated by pre-incubation with superoxide scavenger tempol (1mM) or PEG-superoxide dismutase (100 U/ml). Mean arterial pressure (measured by radiotelemetry), body weight, hepatic steatosis, fasting glucose, glucose tolerance, and insulin sensitivity were similar in HFD-fed KO and WT mice. To identify targets downstream of RBP7, RNA-Sequencing was performed on carotid arteries from 8-week HFD-fed WT and KO mice as well as ND-fed age-matched littermates. Adiponectin (AdipoQ), a PPARγ target, was increased ~6-fold in HFD-fed WT mice, a response that was markedly blunted in KO mice. RNA sequencing was confirmed by qPCR. There was no difference in plasma AdipoQ. AdipoQ protein is expressed in endothelial cells of carotid arteries and its level of expression was increased in HFD-fed WT but not KO mice (AdipoQ/CD31: 1.14±0.1 WT-HFD vs 0.82±0.1 WT-ND, p<0.05; 0.79±0.1 KO-HFD vs 0.81±0.04 KO-ND). This led us to hypothesize that AdipoQ is involved in RBP7-mediated endothelial protection. Incubation of basilar artery with mouse full-length AdipoQ protein (5 μg/mL, 4 hours) significantly ameliorated endothelial dysfunction (ACh, 100 μM: 56±6% AdipoQ+KO vs 26±3% KO, p<0.05) and blunted carotid artery superoxide production in HFD-fed KO mice. AdipoQ also protects against endothelial dysfunction caused by subpressor Ang-II in KO mice. We conclude that RBP7 protects the endothelium from oxidative stress-induced dysfunction through an AdipoQ-dependent mechanism. Our evidence suggests RBP7 is an essential cofactor for activation of some PPARγ target genes in the endothelium.

CTRP3 deficiency reduces liver size and alters IL-6 and TGFβ levels in obese mice.

C1q/TNF-related protein 3 (CTRP3) is a secreted metabolic regulator whose circulating levels are reduced in human and rodent models of obesity and diabetes. Previously, we showed that CTRP3 infusion lowers blood glucose by suppressing gluconeogenesis and that transgenic overexpression of CTRP3 protects mice against diet-induced hepatic steatosis. Here, we used a genetic loss-of-function mouse model to further address whether CTRP3 is indeed required for metabolic homeostasis under normal and obese states. Both male and female mice lacking CTRP3 had similar weight gain when fed a control low-fat (LFD) or high-fat diet (HFD). Regardless of diet, no differences were observed in adiposity, food intake, metabolic rate, energy expenditure, or physical activity levels between wild-type (WT) and Ctrp3-knockout (KO) animals of either sex. Contrary to expectations, loss of CTRP3 in LFD- or HFD-fed male and female mice also had minimal or no impact on whole body glucose metabolism, insulin sensitivity, and fasting-induced hepatic gluconeogenesis. Unexpectedly, the liver sizes of HFD-fed Ctrp3-KO male mice were markedly reduced despite a modest increase in triglyceride content. Furthermore, liver expression of fat oxidation genes was upregulated in the Ctrp3-KO mice. Whereas the liver and adipose expression of profibrotic TGFβ1, as well as its serum levels, was suppressed in HFD-fed KO mice, circulating proinflammatory IL-6 levels were markedly increased; these changes, however, were insufficient to affect systemic metabolic outcome. We conclude that, although it is dispensable for physiological control of energy balance, CTRP3 plays a previously unsuspected role in modulating liver size and circulating cytokine levels in response to obesity.

Modulation of immune-response in female aP2-Cre ERalpha-knockout mice due to high-fat diet feeding and high estradiol levels

Previously reported abnormalities in the reproductive-tract of female adipose-tissue-specific (ap2-Cre) ERα-knockout (ERaKO)-mice were linked with a 40% decrease of ERα-gene-expression in the hypothalamus of these animals, when compared to their control-mice (ERα-floxed;WT). Subsequently, ERaKO-mice showed a pronounced upregulation of estradiol (E2)-plasma levels (KO: 45.9 vs. WT: 19.8pg/ml;p < 0.01) resulting from diminished ERα-mediated feedback in the hypothalamus. When fed with high-fat-diet (HFD), KO-mice developed a pronounced inflammation of uteri. To investigate the cause underlying this inflammatory process we performed H&E-, cd3-, Mac387- and Ly6G-stainings, a semiquantitative scoring of the inflammatory grade and microbiological analysis of the uteri. As HFD-feeding seems to accelerate the inflammatory response in those mice, we also analysed the plasma fatty-acid-profile of the ERaKO-mice fed with a normal-chow-diet (NCD) and HFD. We identified a massive accumulation of neutrophils in the uterus wall, while macrophages and lymphocytes were rarely present. The grade of inflammation (scored from 0 = no inflammation to 3 = most severe form) in HFD-fed KO-mice was significantly higher than in NCD-fed KO-mice (HFD: 2.44 vs. NCD: 1.76 arbitrary units). In accordance, we found that HFD-feeding resulted in increased stearic (c18:0; 983 vs. 567 µg/ml;p < 0.001) and arachidonic (c20:4 n-6; 976 vs. 594 µg/ml;p < 0,001) acid-levels in plasma. Moreover, PCR-analysis of bacterial DNA identified E. coli and Enterococcus sp. as main pathogenic bacteria present in tissue and uterine-fluid of ERaKO-mice. Taken together, our data suggest that high E2-levels in combination with a dysregulated fatty-acid-plasma profile modulate the immune-response of neutrophils and/or macrophages to bacterial infections, resulting in the inability of terminating the inflammatory process.

Abstract 332: Retinol-binding Protein 7 (RBP7) Regulates Endothelial Function, Independent of Metabolic Responses in High-fat Diet Fed Mice

Peroxisome proliferator-activated receptor gamma (PPARG), a ligand-activated transcription factor, plays a protective role in the endothelium through mechanisms that remain unclear. RBP7 is an intracellular retinoid binding protein belonging to the family of fatty acid-binding proteins (FABP) and is a PPARG target. RBP7 expression was increased 2.6-fold by rosiglitazone (RZ), a PPARG agonist in mouse lung endothelial cells, which was blocked by GW9662, a PPARG antagonist. We hypothesized that the protective effects of PPARG on the endothelium are mediated by RBP7. Studies were performed in: 20-week HFD-fed RBP7-deficient (KO) and wild type (WT) and age-matched chow-fed KO and WT mice. HFD increased fasting glucose in both WT and KO mice compared to chow-fed groups, and caused similar hepatic steatosis. HFD-fed mice were more glucose-intolerant and insulin-resistant compared with chow-fed mice, but there were no differences comparing KO with WT mice. Weight gain in response to HFD was also similar in KO and WT mice. Thus KO and WT mice exhibited similar HFD-induced metabolic dysfunction. Endothelium-dependent relaxation to acetylcholine (Ach) was normal in chow-fed KO mice, but was impaired in carotid artery of HFD-fed KO mice (49±6% KO vs 70±4% WT, p&lt;0.05, 30μM Ach). Endothelial-independent relaxation was similar in all groups. Superoxide production in carotid artery (DHE-fluorescence) was elevated 1.5-fold in HFD-fed KO mice compared with the other 3 groups. Normal relaxation to Ach was restored by anti-oxidant treatment, suggesting HFD causes oxidative stress in the absence of RBP7. Systolic blood pressure was similar in HFD-fed KO and WT mice (110±4 mmHg in KO vs 105±0 mmHg in WT). Because other members of the FABP family facilitate steroid hormone receptor function, we hypothesized that RBP7 may facilitate PPARG function. To initially test this, we examined the cellular localization of an RBP7-GFP fusion protein. In vehicle treated COS7 cells, RBP7 was distributed both in cytoplasm and nucleus. RZ treatment resulted in a robust shift of RBP7 into the nucleus. In conclusion, RBP7 protects the endothelium from HFD-induced oxidative and vascular dysfunction, independent of changes to systemic metabolism.

CARD3 Deficiency Exacerbates Diet-Induced Obesity, Hepatosteatosis, and Insulin Resistance in Male Mice

Caspase activation and recruitment domain 3 (CARD3) is a 61-kDa protein kinase with an N-terminal serine/threonine kinase domain and a C-terminal CARD. Previous research on the function of CARD3 has focused on its role in the immune response and inflammatory diseases. Obesity is now a worldwide health problem and is generally recognized as an inflammatory disease. Unexpectedly, we found that CARD3 expression was lower during obesity. In this study, we explored the biological and genetic bases of obesity using CARD3-knockout (KO) and wild-type (WT) mice fed a high-fat diet (HFD) for 24 weeks. We demonstrate that KO mice were more obese than their WT littermates, and KO mice exhibited obvious visceral fat accumulation and liver weight gains after 24 weeks of HFD feeding. We also observed more severe hepatosteatosis in KO mice compared with the WT controls. Hepatic steatosis in the HFD-fed KO mice was linked to a significant increase in the expression of key lipogenic and cholesterol synthesis enzymes, whereas the expression of the enzymes involves in β-oxidation was dramatically reduced. Furthermore, we confirmed the repression of AMP-activated protein kinase signaling and activation of the endoplasmic reticulum stress response. Fatty liver impaired the global glucose and lipid metabolism, which further exacerbated the insulin resistance associated with the repression of Akt signaling and up-regulated systemic inflammation through the M1/M2 (pro- and anti-inflammation) type switch and the activation of the nuclear factor-κB pathway. Our studies demonstrate the crucial role of CARD3 in metabolism and indicate that CARD3 deficiency promotes the diet-induced phenotype of type 2 diabetes.