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Mitochondrial fatty acid oxidation is facilitated by the combined activities of Carnitine Palmitoyltransferase 1 (Cpt1) and Cpt2 which generate and utilize acylcarnitines respectively. We compare the response of mice with liver specific deficiencies in the liver enriched Cpt1a or the ubiquitously expressed Cpt2 and discover that they display unique metabolic, physiological and molecular phenotypes. The loss of Cpt1a or Cpt2 results in the induction of the muscle enriched isoenzyme, Cpt1b, in hepatocytes in a Pparα dependent manner. However, hepatic Cpt1b does not contribute substantively to hepatic fatty acid oxidation when Cpt1a is absent. Both the liver-specific double knockout of Cpt1a and Cpt1b, or Cpt2 eliminates the mitochondrial oxidation of nonesterified fatty acids. However, Cpt1a/Cpt1b double knockout mice retain fatty acid oxidation by utilizing extracellular long chain acylcarnitines that is dependent on Cpt2. These data demonstrate the noncell-autonomous intercellular metabolism of fatty acids in hepatocytes.

To define the metabolic requirements of hematopoiesis, we examined blood lineages in mice conditionally deficient in genes required for long-chain fatty acid oxidation (Cpt2), glutaminolysis (Gls), or mitochondrial pyruvate import (Mpc2). Genetic ablation of Cpt2 or Gls minimally impacted most blood lineages. In contrast, deletion of Mpc2 led to a sharp decline in mature myeloid cells and a slower reduction in T cells, whereas other hematopoietic lineages were unaffected. Yet MPC2-deficient monocytes and neutrophils rapidly recovered due to a transient and specific increase in myeloid progenitor proliferation. Competitive bone marrow chimera and stable isotope tracing experiments demonstrated that this proliferative burst was progenitor intrinsic and accompanied by a metabolic switch to glutaminolysis. Myeloid recovery after loss of MPC2 or cyclophosphamide treatment was delayed in the absence of GLS. Reciprocally, MPC2 was not required for myeloid recovery after cyclophosphamide treatment. Thus, mitochondrial pyruvate metabolism maintains myelopoiesis under steady-state conditions, while glutaminolysis in progenitors promotes emergency myelopoiesis.

Wnt-initiated signaling through a frizzled receptor and the low-density lipoprotein-related receptor-5 coreceptor instructs key anabolic events during skeletal development, homeostasis, and repair. Recent studies indicate that Wnt signaling also regulates the intermediary metabolism of osteoblastic cells, inducing glucose consumption in osteoprogenitors and fatty acid utilization in mature osteoblasts. In this study, we examined the role of the canonical Wnt-signaling target, β-catenin, in the control of osteoblast metabolism. In vitro, Wnt ligands and agonists that stimulated β-catenin activation in osteoblasts enhanced fatty acid catabolism, whereas genetic ablation of β-catenin dramatically reduced oleate oxidation concomitant with reduced osteoblast maturation and increased glycolytic metabolism. Temporal ablation of β-catenin expression in osteoblasts in vivo produced the expected low-bone-mass phenotype and also led to an increase in white adipose tissue mass, dyslipidemia, and impaired insulin sensitivity. Because the expression levels of enzymatic mediators of fatty acid β-oxidation are reduced in the skeleton of β-catenin mutants, these results further confirm the role of the osteoblast in lipid metabolism and indicate that the influence of Wnt signaling on fatty acid utilization proceeds via its canonical signaling pathway.

The liver has a large capacity for mitochondrial fatty acid β-oxidation, which is critical for systemic metabolic adaptations such as gluconeogenesis and ketogenesis. To understand the role of hepatic fatty acid oxidation in response to a chronic high-fat diet (HFD), we generated mice with a liver-specific deficiency of mitochondrial long-chain fatty acid β-oxidation (Cpt2L−/−mice). Paradoxically, Cpt2L−/−mice were resistant to HFD-induced obesity and glucose intolerance with an absence of liver damage, although they exhibited serum dyslipidemia, hepatic oxidative stress, and systemic carnitine deficiency. Feeding an HFD induced hepatokines in mice, with a loss of hepatic fatty acid oxidation that enhanced systemic energy expenditure and suppressed adiposity. Additionally, the suppression in hepatic gluconeogenesis was sufficient to improve HFD-induced glucose intolerance. These data show that inhibiting hepatic fatty acid oxidation results in a systemic hormetic response that protects mice from HFD-induced obesity and glucose intolerance.

Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥

Recent studies have identified the osteoblast as an insulin responsive cell that participates in global energy homeostasis. Here, we show that glucose transporter-4 (Glut4) is required for insulin-dependent uptake and oxidation of glucose in mature osteoblasts. In primary cultures of mouse osteoblasts, insulin increased uptake and oxidation of 14C-glucose in a dose-dependent fashion but did not significantly affect uptake or oxidation of 14C-oleate. In vitro, undifferentiated osteoblasts expressed 3 high-affinity Gluts: Glut1, Glut4, and Glut3. However, although levels of Glut1 and Glut3 remained constant during the course of osteoblast differentiation, Glut4 expression increased by 5-fold in association with enhanced insulin-stimulated glucose uptake. Glut4 ablation in osteoblasts in vitro eliminated insulin-stimulated glucose uptake, reduced proliferation and diminished measures of osteoblast maturation. In vivo, Glut4 expression was observed in osteoblasts, osteocytes, and chondrocytes at a level approaching that observed in adjacent skeletal muscle. To determine the importance of Glut4 in bone in vivo, we generated mice lacking Glut4 in osteoblasts and osteocytes (ΔGlut4). ΔGlut4 mice exhibited normal bone architecture but exhibited an increase in peripheral fat in association with hyperinsulinemia, β-cell islet hypertrophy, and reduced insulin sensitivity. Surprisingly, the expression of insulin target genes in liver, muscle, and adipose from ΔGlut4 mice were unchanged or increased, indicating that alterations in glucose homeostasis were the result of reduced clearance by bone. These findings suggest that Glut4 mediates insulin-stimulated glucose uptake by mature osteoblasts/osteocytes and that the magnitude of glucose use by bone cells is sufficient to impact global glucose disposal in the mouse.

The liver is critical for maintaining systemic energy balance during starvation. To understand the role of hepatic fatty acid β-oxidation on this process, we generated mice with a liver-specific knockout of carnitine palmitoyltransferase 2 (Cpt2L−/−), an obligate step in mitochondrial long-chain fatty acid β-oxidation. Fasting induced hepatic steatosis and serum dyslipidemia with an absence of circulating ketones, while blood glucose remained normal. Systemic energy homeostasis was largely maintained in fasting Cpt2L−/−mice by adaptations in hepatic and systemic oxidative gene expression mediated in part by Pparα target genes including procatabolic hepatokines Fgf21, Gdf15, and Igfbp1. Feeding a ketogenic diet to Cpt2L−/−mice resulted in severe hepatomegaly, liver damage, and death with a complete absence of adipose triglyceride stores. These data show that hepatic fatty acid oxidation is not required for survival during acute food deprivation but essential for constraining adipocyte lipolysis and regulating systemic catabolism when glucose is limiting.

Ambient temperature affects energy intake and expenditure to maintain homeostasis in a continuously fluctuating environment. Here, mice with an adipose-specific defect in fatty acid oxidation (Cpt2A−/−) were subjected to varying temperatures to determine the role of adipose bioenergetics in environmental adaptation and body weight regulation. Microarray analysis of mice acclimatized to thermoneutrality revealed that Cpt2A−/−interscapular brown adipose tissue (BAT) failed to induce the expression of thermogenic genes such as Ucp1and Pgc1αin response to adrenergic stimulation, and increasing ambient temperature exacerbated these defects. Furthermore, thermoneutral housing induced mtDNA stress in Cpt2A−/−BAT and ultimately resulted in a loss of interscapular BAT. Although the loss of adipose fatty acid oxidation resulted in clear molecular, cellular, and physiologic deficits in BAT, body weight gain and glucose tolerance were similar in control and Cpt2A−/−mice in response to a high-fat diet, even when mice were housed at thermoneutrality.

Upon antigen recognition and co-stimulation, T lymphocytes upregulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic reprogramming in T cells regulates T cell activation and differentiation but is not just a consequence of antigen recognition. Although such metabolic reprogramming promotes the differentiation and function of T effector cells, the differentiation of regulatory T cells employs different metabolic reprogramming. Therefore, we hypothesized that inhibition of glycolysis and glutamine metabolism might prevent graft rejection by inhibiting effector generation and function and promoting regulatory T cell generation. We devised an anti-rejection regimen involving the glycolytic inhibitor 2-deoxyglucose (2-DG), the anti-type II diabetes drug metformin, and the inhibitor of glutamine metabolism 6-diazo-5-oxo-L-norleucine (DON). Using this triple-drug regimen, we were able to prevent or delay graft rejection in fully mismatched skin and heart allograft transplantation models.

To understand the contribution of adipose tissue fatty acid oxidation to whole-body metabolism, we generated mice with an adipose-specific knockout of carnitine palmitoyltransferase 2 (CPT2A−/−), an obligate step in mitochondrial long-chain fatty acid oxidation. CPT2A−/−mice became hypothermic after an acute cold challenge, and CPT2A−/−brown adipose tissue (BAT) failed to upregulate thermogenic genes in response to agonist-induced stimulation. The adipose-specific loss of CPT2 resulted in diet-dependent changes in adiposity but did not result in changes in body weight on low- or high-fat diets. Additionally, CPT2A−/−mice had suppressed high-fat diet-induced oxidative stress and inflammation in visceral white adipose tissue (WAT); however, high-fat diet-induced glucose intolerance was not improved. These data show that fatty acid oxidation is required for cold-induced thermogenesis in BAT and high-fat diet-induced oxidative stress and inflammation in WAT.

Endothelial cells (ECs) are believed to be an important component in the protection from lipopolysaccharide (LPS)-induced endotoxic shock. However, the cellular and molecular mechanism is not well defined. Here, we report that signal transducer and activator of transcription (STAT) 3 is an essential regulator of the antiinflammatory function of ECs in systemic immunity. Because STAT3 deficiency results in early embryonic lethality, we have generated mice with a conditional STAT3 deletion in endothelium (STAT3E−/−). STAT3E−/− mice are healthy and fertile, and isolated ECs initiate normal tube formation in vitro. Conditional endothelial but not organ-specific (i.e., hepatocyte or cardiomyocyte) STAT3 knockout mice show an increased susceptibility to lethality after LPS challenge. The LPS response in STAT3E−/− mice shows exaggerated inflammation and leukocyte infiltration in multiple organs combined with elevated activity of serum alanine aminotransferase and aspartate aminotransferase, indicating organ damage. Concomitantly, proinflammatory cytokines are produced at an exaggerated level and for a prolonged period. This defect cannot be explained by lack of antiinflammatory cytokines, such as interleukin 10 and transforming growth factor β. Instead, we have shown that a soluble activity derived from endothelia and dependent on STAT3 is critical for suppression of interferon γ. These data define STAT3 signaling within endothelia as a critical antiinflammatory mediator and provide new insight to the protective function of ECs in inflammation.

Odontocete cetaceans exhibit genomic mutations in key ketogenesis genes. In order to validate an inferred lack of ketogenesis made by observations from genome sequencing, we biochemically analyzed tissues from several odontocete cetacean species and demonstrate that they indeed do not exhibit appreciable hepatic β-hydroxybutyrate (βHB) or its carnitine ester. Furthermore, liver tissue exhibited significantly lower long chain acylcarnitines and increased odd chain acylcarnitines indicative of a decreased reliance on hepatic long chain fatty acid oxidation in these carnivorous mammals. Finally, we performed single molecule, real-time next generation sequencing of liver and brain RNA of Tursiops truncatus and demonstrate that the succinyl-CoA transferase required for acetoacetate catabolism is expressed in the nervous system. These data show that odontocete cetaceans have lost the ability to perform ketogenesis and suggest a hepatocentric coenzyme A recycling function rather than a predominantly systemic–bioenergetic role for ketogenesis in other ketogenic competent mammals such as humans.

Mitochondrial carriers (MCs) mediate the passage of small molecules across the inner mitochondrial membrane (IMM), enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterize SFXN1, a member of the non-canonical, sideroflexin family. We find that SFXN1, an integral IMM protein with an uneven number of transmembrane domains, is a TIM22 complex substrate. SFXN1 deficiency leads to mitochondrial respiratory chain impairments, most detrimental to complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. The CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and α-ketoglutarate metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency.

The extreme metabolic demands of pregnancy require coordinated metabolic adaptations between mother and fetus to balance fetal growth and maternal health with nutrient availability. To determine maternal and fetal contributions to metabolic flexibility during gestation, pregnant mice with genetic impairments in mitochondrial carbohydrate and/or lipid metabolism were subjected to nutrient deprivation. The maternal fasting response initiates a fetal liver transcriptional program marked by upregulation of lipid- and peroxisome proliferator-activated receptor alpha (Pparα)-regulated genes. Impaired maternal lipid metabolism alters circulating lipid metabolite concentrations and enhances the fetal response to fasting, which is largely dependent on fetal Pparα. Maternal fasting also improves metabolic deficits in fetal carbohydrate metabolism by increasing the availability of alternative substrates. Impairment of both carbohydrate and lipid metabolism in pregnant dams further exacerbates the fetal liver transcriptional response to nutrient deprivation. Together, these data demonstrate a regulatory role for mitochondrial macronutrient metabolism in mediating maternal-fetal metabolic communication, particularly when nutrients are limited.