LipidFinder: A computational workflow for discovery of lipids identifies eicosanoid-phosphoinositides in platelets

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Abstract

Accurate and high-quality curation of lipidomic datasets generated from plasma, cells, or tissues is becoming essential for cell biology investigations and biomarker discovery for personalized medicine. However, a major challenge lies in removing artifacts otherwise mistakenly interpreted as real lipids from large mass spectrometry files (>60 K features), while retaining genuine ions in the dataset. This requires powerful informatics tools; however, available workflows have not been tailored specifically for lipidomics, particularly discovery research. We designed LipidFinder, an open-source Python workflow. An algorithm is included that optimizes analysis based on users’ own data, and outputs are screened against online databases and categorized into LIPID MAPS classes. LipidFinder outperformed three widely used metabolomics packages using data from human platelets. We show a family of three 12-hydroxyeicosatetraenoic acid phosphoinositides (16:0/, 18:1/, 18:0/12-HETE-PI) generated by thrombin-activated platelets, indicating crosstalk between eicosanoid and phosphoinositide pathways in human cells. The software is available on GitHub (https://github.com/cjbrasher/LipidFinder), with full userguides.

Authors

Anne O’Connor, Christopher J. Brasher, David A. Slatter, Sven W. Meckelmann, Jade I. Hawksworth, Stuart M. Allen, Valerie B. O’Donnell

Insulin’s direct hepatic effect explains the inhibition of glucose production caused by insulin secretion

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Abstract

Insulin can inhibit hepatic glucose production (HGP) by acting directly on the liver as well as indirectly through effects on adipose tissue, pancreas, and brain. While insulin’s indirect effects are indisputable, their physiologic role in the suppression of HGP seen in response to increased insulin secretion is not clear. Likewise, the mechanisms by which insulin suppresses lipolysis and pancreatic α cell secretion under physiologic circumstances are also debated. In this study, insulin was infused into the hepatic portal vein to mimic increased insulin secretion, and insulin’s indirect liver effects were blocked either individually or collectively. During physiologic hyperinsulinemia, plasma free fatty acid (FFA) and glucagon levels were clamped at basal values and brain insulin action was blocked, but insulin’s direct effects on the liver were left intact. Insulin was equally effective at suppressing HGP when its indirect effects were absent as when they were present. In addition, the inhibition of lipolysis, as well as glucagon and insulin secretion, did not require CNS insulin action or decreased plasma FFA. This indicates that the rapid suppression of HGP is attributable to insulin’s direct effect on the liver and that its indirect effects are redundant in the context of a physiologic increase in insulin secretion.

Authors

Dale S. Edgerton, Guillaume Kraft, Marta Smith, Ben Farmer, Phillip E. Williams, Katie C. Coate, Richard L. Printz, Richard M. O’Brien, Alan D. Cherrington

Liver X receptor α mediates hepatic triglyceride accumulation through upregulation of G0/G1 Switch Gene 2 expression

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Abstract

Liver X receptors (LXRs) are transcription factors essential for cholesterol homeostasis and lipogenesis. LXRα has been implicated in regulating hepatic triglyceride (TG) accumulation upon both influx of adipose-derived fatty acids (FAs) during fasting and stimulation of de novo FA synthesis by chemical agonism of LXR. However, whether or not a convergent mechanism is employed to drive deposition of FAs from these 2 different sources in TGs is undetermined. Here, we report that the G0/G1 Switch Gene 2 (G0S2), a selective inhibitor of intracellular TG hydrolysis/lipolysis, is a direct target gene of LXRα. Transcriptional activation is conferred by LXRα binding to a direct repeat 4 (DR4) motif in the G0S2 promoter. While LXRα^–/– mice exhibited decreased hepatic G0S2 expression, adenoviral expression of G0S2 was sufficient to restore fasting-induced TG storage and glycogen depletion in the liver of these mice. In response to LXR agonist T0901317, G0S2 ablation prevented hepatic steatosis and hypertriglyceridemia without affecting the beneficial effects on HDL. Thus, the LXRα-G0S2 axis plays a distinct role in regulating hepatic TG during both fasting and pharmacological activation of LXR.

Authors

Bradlee L. Heckmann, Xiaodong Zhang, Alicia M. Saarinen, Gabriele Schoiswohl, Erin E. Kershaw, Rudolf Zechner, Jun Liu

Switching harmful visceral fat to beneficial energy combustion improves metabolic dysfunctions

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Abstract

Visceral fat is considered the genuine and harmful white adipose tissue (WAT) that is associated to development of metabolic disorders, cardiovascular disease, and cancer. Here, we present a new concept to turn the harmful visceral fat into a beneficial energy consumption depot, which is beneficial for improvement of metabolic dysfunctions in obese mice. We show that low temperature–dependent browning of visceral fat caused decreased adipose weight, total body weight, and body mass index, despite increased food intake. In high-fat diet–fed mice, low temperature exposure improved browning of visceral fat, global metabolism via nonshivering thermogenesis, insulin sensitivity, and hepatic steatosis. Genome-wide expression profiling showed upregulation of WAT browning–related genes including Cidea and Dio2. Conversely, Prdm16 was unchanged in healthy mice or was downregulated in obese mice. Surgical removal of visceral fat and genetic knockdown of UCP1 in epididymal fat largely ablated low temperature–increased global thermogenesis and resulted in the death of most mice. Thus, browning of visceral fat may be a compensatory heating mechanism that could provide a novel therapeutic strategy for treating visceral fat–associated obesity and diabetes.

Authors

Xiaoyan Yang, Wenhai Sui, Meng Zhang, Mei Dong, Sharon Lim, Takahiro Seki, Ziheng Guo, Carina Fischer, Huixia Lu, Cheng Zhang, Jianmin Yang, Meng Zhang, Yangang Wang, Caixia Cao, Yanyan Gao, Xingguo Zhao, Meili Sun, Yuping Sun, Rujie Zhuang, Nilesh J. Samani, Yun Zhang, Yihai Cao

The human brain produces fructose from glucose

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Abstract

Fructose has been implicated in the pathogenesis of obesity and type 2 diabetes. In contrast to glucose, CNS delivery of fructose in rodents promotes feeding behavior. However, because circulating plasma fructose levels are exceedingly low, it remains unclear to what extent fructose crosses the blood-brain barrier to exert CNS effects. To determine whether fructose can be endogenously generated from glucose via the polyol pathway (glucose → sorbitol → fructose) in human brain, 8 healthy subjects (4 women/4 men; age, 28.8 ± 6.2 years; BMI, 23.4 ± 2.6; HbA1C, 4.9% ± 0.2%) underwent ¹H magnetic resonance spectroscopy scanning to measure intracerebral glucose and fructose levels during a 4-hour hyperglycemic clamp (plasma glucose, 220 mg/dl). Using mixed-effects regression model analysis, intracerebral glucose rose significantly over time and differed from baseline at 20 to 230 minutes. Intracerebral fructose levels also rose over time, differing from baseline at 30 to 230 minutes. The changes in intracerebral fructose were related to changes in intracerebral glucose but not to plasma fructose levels. Our findings suggest that the polyol pathway contributes to endogenous CNS production of fructose and that the effects of fructose in the CNS may extend beyond its direct dietary consumption.

Authors

Janice J. Hwang, Lihong Jiang, Muhammad Hamza, Feng Dai, Renata Belfort-DeAguiar, Gary Cline, Douglas L. Rothman, Graeme Mason, Robert S. Sherwin

Foxp3 drives oxidative phosphorylation and protection from lipotoxicity

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Abstract

Tregs can adopt a catabolic metabolic program with increased capacity for fatty acid oxidation–fueled oxidative phosphorylation (OXPHOS). It is unclear why this form of metabolism is favored in Tregs and, more specifically, whether this program represents an adaptation to the environment and developmental cues or is “hardwired” by Foxp3. Here we show, using metabolic analysis and an unbiased mass spectroscopy–based proteomics approach, that Foxp3 is both necessary and sufficient to program Treg-increased respiratory capacity and Tregs’ increased ability to utilize fatty acids to fuel oxidative phosphorylation. Foxp3 drives upregulation of components of all the electron transport complexes, increasing their activity and ATP generation by oxidative phosphorylation. Increased fatty acid β-oxidation also results in selective protection of Foxp3⁺ cells from fatty acid–induced cell death. This observation may provide novel targets for modulating Treg function or selection therapeutically.

Authors

Duncan Howie, Stephen Paul Cobbold, Elizabeth Adams, Annemieke Ten Bokum, Andra Stefania Necula, Wei Zhang, Honglei Huang, David J. Roberts, Benjamin Thomas, Svenja S. Hester, David J. Vaux, Alexander G. Betz, Herman Waldmann

Gα₁₁ mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

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Abstract

Heterozygous germline gain-of-function mutations of G-protein subunit α₁₁ (Gα₁₁), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) — which has a germline hypermorphic Gα₁₁ mutation, Ile62Val — may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα₁₁ to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα₁₁ mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.

Authors

Caroline M. Gorvin, Fadil M. Hannan, Sarah A. Howles, Valerie N. Babinsky, Sian E. Piret, Angela Rogers, Andrew J. Freidin, Michelle Stewart, Anju Paudyal, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Tonia L. Vincent, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker

Mitochondrial quality-control dysregulation in conditional HO-1^–/– mice

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Abstract

The heme oxygenase-1 (Hmox1; HO-1) pathway was tested for defense of mitochondrial quality control in cardiomyocyte-specific Hmox1 KO mice (HO-1[CM]^–/–) exposed to oxidative stress (100% O₂). After 48 hours of exposure, these mice showed persistent cardiac inflammation and oxidative tissue damage that caused sarcomeric disruption, cardiomyocyte death, left ventricular dysfunction, and cardiomyopathy, while control hearts showed minimal damage. After hyperoxia, HO-1(CM)^–/– hearts showed suppression of the Pgc-1α/nuclear respiratory factor-1 (NRF-1) axis, swelling, low electron density mitochondria by electron microscopy (EM), increased cell death, and extensive collagen deposition. The damage mechanism involves structurally deficient autophagy/mitophagy, impaired LC3II processing, and failure to upregulate Pink1- and Park2-mediated mitophagy. The mitophagy pathway was suppressed through loss of NRF-1 binding to proximal promoter sites on both genes. These results indicate that cardiac Hmox1 induction not only prevents heme toxicity, but also regulates the timing and registration of genetic programs for mitochondrial quality control that limit cell death, pathological remodeling, and cardiac fibrosis.

Authors

Hagir B. Suliman, Jeffrey E. Keenan, Claude A. Piantadosi

Redirecting N-acetylaspartate metabolism in the central nervous system normalizes myelination and rescues Canavan disease

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Abstract

Canavan disease (CD) is a debilitating and lethal leukodystrophy caused by mutations in the aspartoacylase (ASPA) gene and the resulting defect in N-acetylaspartate (NAA) metabolism in the CNS and peripheral tissues. Recombinant adeno-associated virus (rAAV) has the ability to cross the blood-brain barrier and widely transduce the CNS. We developed a rAAV-based and optimized gene replacement therapy, which achieves early, complete, and sustained rescue of the lethal disease phenotype in CD mice. Our treatment results in a super-mouse phenotype, increasing motor performance of treated CD mice beyond that of WT control mice. We demonstrate that this rescue is oligodendrocyte independent, and that gene correction in astrocytes is sufficient, suggesting that the establishment of an astrocyte-based alternative metabolic sink for NAA is a key mechanism for efficacious disease rescue and the super-mouse phenotype. Importantly, the use of clinically translatable high-field imaging tools enables the noninvasive monitoring and prediction of therapeutic outcomes for CD and might enable further investigation of NAA-related cognitive function.

Authors

Dominic J. Gessler, Danning Li, Hongxia Xu, Qin Su, Julio Sanmiguel, Serafettin Tuncer, Constance Moore, Jean King, Reuben Matalon, Guangping Gao

Adipocyte JAK2 mediates growth hormone–induced hepatic insulin resistance

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Abstract

For nearly 100 years, growth hormone (GH) has been known to affect insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here, we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extrahepatic, adipose tissue–dependent mechanism.

Authors

Kevin C. Corbit, João Paulo G. Camporez, Jennifer L. Tran, Camella G. Wilson, Dylan A. Lowe, Sarah M. Nordstrom, Kirthana Ganeshan, Rachel J. Perry, Gerald I. Shulman, Michael J. Jurczak, Ethan J. Weiss