The metabolic enzyme pyruvate kinase M2 regulates platelet function and arterial thrombosis.

Very little is known about the role of metabolic regulatory mechanisms in platelet activation and thrombosis. Dimeric pyruvate kinase M2 (PKM2) is a crucial regulator of aerobic glycolysis that facilitates the production of lactate and metabolic reprogramming. Herein, we report that limiting PKM2 dimer formation, using the small molecule inhibitor ML265, negatively regulates lactate production and glucose uptake in human and murine stimulated platelets. Furthermore, limiting PKM2 dimer formation reduced agonist-induced platelet activation, aggregation, clot retraction, and thrombus formation under arterial shear stress in vitro in both human and murine platelets. Mechanistically, limiting PKM2 dimerization downregulated phosphatidylinositol 3-kinase (PI3K)-mediated protein kinase B or serine/threonine-specific protein kinase (Akt)/glycogen synthase kinase 3 (GSK3) signaling in human and murine platelets. To provide further evidence for the role of PKM2 in platelet function, we generated a megakaryocyte or platelet-specific PKM2-/- mutant strain (PKM2fl/flPF4Cre+). Platelet-specific PKM2-deficient mice exhibited impaired agonist-induced platelet activation, aggregation, clot retraction, and PI3K-mediated Akt/GSK3 signaling and were less susceptible to arterial thrombosis in FeCl3 injury-induced carotid- and laser injury-induced mesenteric artery thrombosis models, without altering hemostasis. Wild-type mice treated with ML265 were less susceptible to arterial thrombosis with unaltered tail bleeding times. These findings reveal a major role for PKM2 in coordinating multiple aspects of platelet function, from metabolism to cellular signaling to thrombosis, and implicate PKM2 as a potential target for antithrombotic therapeutic intervention.

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects.

Adipose TBX1 regulates ß-adrenergic sensitivity in subcutaneous adipose tissue and thermogenic capacity in vivo.

OBJECTIVE: T-box 1 (TBX1) has been identified as a genetic marker of beige adipose tissue. TBX1 is a mesodermal development transcription factor essential for tissue patterning and cell fate determination. However, whether it plays a role in the process of adipose beiging or how it functions in adipose tissue has not been reported. Here, we examined the function of TBX1 in adipose tissue as well as adipose-derived stem cells from mice and humans. METHODS: Adipose-specific TBX1 transgenic (TBX1 AdipoTG) and adipose-specific TBX1 knockout (TBX1 AdipoKO) mice were generated to explore the function of TBX1 in the process of adipose beiging, metabolism and energy homeostasis in vivo. In vitro, we utilized a siRNA mediated approach to determine the function of TBX1 during adipogenesis in mouse and human stem cells. RESULTS: Adipose-specific overexpression of TBX1 was not sufficient to fully induce beiging and prevent diet-induced obesity. However, adipose TBX1 expression was necessary to defend body temperature during cold through regulation of UCP1 and for maintaining ß3-adrenergic sensitivity and glucose homeostasis in vivo. Loss of adipose TBX1 expression enhanced basal lipolysis and reduced the size of subcutaneous iWAT adipocytes. Reduction of TBX1 expression via siRNA significantly impaired adipogenesis of mouse stromal vascular cells but significantly enhanced adipogenesis in human adipose derived stem cells. CONCLUSIONS: Adipose expression of TBX1 is necessary, but not sufficient, to defend body temperature during cold via proper UCP1 expression. Adipose TBX1 expression was also required for proper insulin signaling in subcutaneous adipose as well as for maintaining ß-adrenergic sensitivity, but overexpression of TBX1 was not sufficient to induce adipocyte beiging or to prevent diet-induced obesity. TBX1 expression is enriched in adipose stem cells in which it has contrasting effects on adipogenesis in mouse versus human cells. Collectively, these data demonstrate the importance of adipose TBX1 in the regulation of beige adipocyte function, energy homeostasis, and adipocyte development.

Liver Derived FGF21 Maintains Core Body Temperature During Acute Cold Exposure.

Fibroblast Growth Factor 21 (FGF21) elicits an array of metabolic effects. However, the physiological role of FGF21 during thermal challenges is not clear. In this study, we assessed the tissue source of FGF21 and its site of action to regulate core body temperature in response to cold. Using mice lacking FGF21 specifically in the liver (FGF21 LivKO) or adipose tissues (FGF21 AdipoKO), we performed a series of cold exposure studies to examine the tissue specific induction of FGF21 in response to cold. We also examined the physiological site of FGF21 action during cold exposure by impairing FGF21 signaling to adipose tissues or the central nervous system (CNS) using genetic ablation of the FGF21 co-receptor ß-klotho in adipose tissues (KLB AdipoKO) or pharmacological blockage of FGF21 signaling. We found that only liver-derived FGF21 enters circulation during acute cold exposure and is critical for thermoregulation. While FGF21 signaling directly to adipose tissues during cold is dispensable for thermoregulation, central FGF21 signaling is necessary for maximal sympathetic drive to brown adipose tissue to maintain thermoregulation during cold. These data demonstrate a previously unrecognized role for FGF21 in the maintenance of body temperature in response to cold.

Defining "FGF21 Resistance" during obesity: Controversy, criteria and unresolved questions.

The term "FGF21 resistance" was first used to describe increased circulating FGF21 levels concomitant to decreased FGF21 receptor complex expression in white adipose tissue of obese mice.  Since this initial report, the term has been associated with a wide range of pathological states, including human obesity, in which circulating FGF21 levels are elevated. However, the notion of "FGF21 resistance" has been controversial partly due to difficulty in delineating the mechanisms underlying the physiological versus pharmacological effects of FGF21.  Here, key aspects of the term "FGF21 resistance" are discussed including; the origin and experimental context surrounding the term "FGF21 resistance", new criteria for evaluating FGF21 sensitivity in vivo and finally, crucial unresolved questions regarding the function of FGF21 during obesity.

Scaffold-free generation of uniform adipose spheroids for metabolism research and drug discovery.

Adipose tissue dysfunction is critical to the development of type II diabetes and other metabolic diseases. While monolayer cell culture has been useful for studying fat biology, 2D culture often does not reflect the complexity of fat tissue. Animal models are also problematic in that they are expensive, time consuming, and may not completely recapitulate human biology because of species variation. To address these problems, we have developed a scaffold-free method to generate 3D adipose spheroids from primary or immortal human or mouse pre-adipocytes. Pre-adipocytes self-organize into spheroids in hanging drops and upon transfer to low attachment plates, can be maintained in long-term cultures. Upon exposure to differentiation cues, the cells mature into adipocytes, accumulating large lipid droplets that expand with time. The 3D spheroids express and secrete higher levels of adiponectin compared to 2D culture and respond to stress, either culture-related or toxin-associated, by secreting pro-inflammatory adipokines. In addition, 3D spheroids derived from brown adipose tissue (BAT) retain expression of BAT markers better than 2D cultures derived from the same tissue. Thus, this model can be used to study both the maturation of pre-adipocytes or the function of mature adipocytes in a 3D culture environment.

FGF21 resistance is not mediated by downregulation of beta-klotho expression in white adipose tissue.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates metabolic homeostasis. Previous work has suggested that impairment of FGF21 signaling in adipose tissue may occur through downregulation of the obligate FGF21 co-receptor, ß-klotho, which leads to "FGF21 resistance" during the onset of diet-induced obesity. Here, we sought to determine whether maintenance of ß-klotho expression in adipose tissue prevents FGF21 resistance and whether other mechanisms also contribute to FGF21 resistance in vivo. METHODS: We generated adipose-specific ß-klotho transgenic mice to determine whether maintenance of ß-klotho expression in adipose tissue prevents FGF21 resistance in vivo. RESULTS: ß-klotho protein levels are markedly decreased in white adipose tissue, but not liver or brown adipose tissue, during diet-induced obesity. Maintenance of ß-klotho protein expression in adipose tissue does not alleviate impaired FGF21 signaling in white adipose or increase FGF21 sensitivity in vivo. CONCLUSIONS: In white adipose tissue, downregulation of ß-klotho expression is not the major mechanism contributing to impaired FGF21 signaling in white adipose tissue.

FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms.

FGF21 is an endocrine hormone that regulates energy homeostasis and insulin sensitivity. The mechanism of FGF21 action and the tissues responsible for these effects have been controversial, with both adipose tissues and the central nervous system having been identified as the target site mediating FGF21-dependent increases in insulin sensitivity, energy expenditure, and weight loss. Here we show that, while FGF21 signaling to adipose tissue is required for the acute insulin-sensitizing effects of FGF21, FGF21 signaling to adipose tissue is not required for its chronic effects to increase energy expenditure and lower body weight. Also, in contrast to previous studies, we found that adiponectin is dispensable for the metabolic effects of FGF21 in increasing insulin sensitivity and energy expenditure. Instead, FGF21 acutely enhances insulin sensitivity through actions on brown adipose tissue. Our data reveal that the acute and chronic effects of FGF21 can be dissociated through adipose-dependent and -independent mechanisms.

Obesity-associated NLRC4 inflammasome activation drives breast cancer progression.

Obesity is associated with an increased risk of developing breast cancer and is also associated with worse clinical prognosis. The mechanistic link between obesity and breast cancer progression remains unclear, and there has been no development of specific treatments to improve the outcome of obese cancer patients. Here we show that obesity-associated NLRC4 inflammasome activation/ interleukin (IL)-1 signalling promotes breast cancer progression. The tumour microenvironment in the context of obesity induces an increase in tumour-infiltrating myeloid cells with an activated NLRC4 inflammasome that in turn activates IL-1ß, which drives disease progression through adipocyte-mediated vascular endothelial growth factor A (VEGFA) expression and angiogenesis. Further studies show that treatment of mice with metformin inhibits obesity-associated tumour progression associated with a marked decrease in angiogenesis. This report provides a causal mechanism by which obesity promotes breast cancer progression and lays out a foundation to block NLRC4 inflammasome activation or IL-1ß signalling transduction that may be useful for the treatment of obese cancer patients.

Suppression of Resting Metabolism by the Angiotensin AT2 Receptor.

Activation of the brain renin-angiotensin system (RAS) stimulates energy expenditure through increasing of the resting metabolic rate (RMR), and this effect requires simultaneous suppression of the circulating and/or adipose RAS. To identify the mechanism by which the peripheral RAS opposes RMR control by the brain RAS, we examined mice with transgenic activation of the brain RAS (sRA mice). sRA mice exhibit increased RMR through increased energy flux in the inguinal adipose tissue, and this effect is attenuated by angiotensin II type 2 receptor (AT2) activation. AT2 activation in inguinal adipocytes opposes norepinephrine-induced uncoupling protein-1 (UCP1) production and aspects of cellular respiration, but not lipolysis. AT2 activation also opposes inguinal adipocyte function and differentiation responses to epidermal growth factor (EGF). These results highlight a major, multifaceted role for AT2 within inguinal adipocytes in the control of RMR. The AT2 receptor may therefore contribute to body fat distribution and adipose depot-specific effects upon cardio-metabolic health.

Metabolic fibroblast growth factors (FGFs): Mediators of energy homeostasis.

The metabolic fibroblast growth factors (FGFs), FGF1, FGF15/19, and FGF21 differ from classic FGFs in that they modulate energy homeostasis in response to fluctuating nutrient availability. These unique mediators of metabolism regulate a number of physiological processes which contribute to their potent pharmacological properties. Administration of pharmacological doses of these FGFs causes weight loss, increases energy expenditure, and improves carbohydrate and lipid metabolism in obese animal models. However, many questions remain regarding the precise molecular and physiological mechanisms governing the effects of individual metabolic FGFs. Here we review the metabolic actions of FGF1, FGF15/19, and FGF21 while providing insights into their pharmacological effects by examining known biological functions.

Central serotonergic neurons activate and recruit thermogenic brown and beige fat and regulate glucose and lipid homeostasis.

Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids. Serotonin (5-HT) neurons in the CNS are essential for thermoregulation and accordingly may control metabolic activity of thermogenic fat. To test this, we generated mice in which the human diphtheria toxin receptor (DTR) was selectively expressed in central 5-HT neurons. Treatment with diphtheria toxin (DT) eliminated 5-HT neurons and caused loss of thermoregulation, brown adipose tissue (BAT) steatosis, and a >50% decrease in uncoupling protein 1 (Ucp1) expression in BAT and inguinal white adipose tissue (WAT). In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold, and triglycerides 6.5-fold. Similar BAT and beige fat defects occurred in Lmx1b(f/f)ePet1(Cre) mice in which 5-HT neurons fail to develop in utero. We conclude 5-HT neurons play a major role in regulating glucose and lipid homeostasis, in part through recruitment and metabolic activation of brown and beige adipocytes.

A novel role for subcutaneous adipose tissue in exercise-induced improvements in glucose homeostasis.

Exercise training improves whole-body glucose homeostasis through effects largely attributed to adaptations in skeletal muscle; however, training also affects other tissues, including adipose tissue. To determine whether exercise-induced adaptations to adipose tissue contribute to training-induced improvements in glucose homeostasis, subcutaneous white adipose tissue (scWAT) from exercise-trained or sedentary donor mice was transplanted into the visceral cavity of sedentary recipients. Remarkably, 9 days post-transplantation, mice receiving scWAT from exercise-trained mice had improved glucose tolerance and enhanced insulin sensitivity compared with mice transplanted with scWAT from sedentary or sham-treated mice. Mice transplanted with scWAT from exercise-trained mice had increased insulin-stimulated glucose uptake in tibialis anterior and soleus muscles and brown adipose tissue, suggesting that the transplanted scWAT exerted endocrine effects. Furthermore, the deleterious effects of high-fat feeding on glucose tolerance and insulin sensitivity were completely reversed if high-fat-fed recipient mice were transplanted with scWAT from exercise-trained mice. In additional experiments, voluntary exercise training by wheel running for only 11 days resulted in profound changes in scWAT, including the increased expression of ∼1,550 genes involved in numerous cellular functions including metabolism. Exercise training causes adaptations to scWAT that elicit metabolic improvements in other tissues, demonstrating a previously unrecognized role for adipose tissue in the beneficial effects of exercise on systemic glucose homeostasis.

Circulating FGF21 is liver derived and enhances glucose uptake during refeeding and overfeeding.

Fibroblast growth factor (FGF)21 is an endocrine hormone that is expressed in multiple tissues and functions physiologically to maintain energy homeostasis. FGF21 is being pursued as a therapeutic target for diabetes and obesity because of its rapid and potent effects on improving insulin sensitivity. However, whether FGF21 enhances insulin sensitivity under physiologic conditions remains unclear. Here, we show that liver-derived FGF21 enters the circulation during fasting but also remains present and functional during the early stage of refeeding. After a prolonged fast, FGF21 acts as an insulin sensitizer to overcome the peripheral insulin resistance induced by fasting, thereby maximizing glucose uptake. Likewise, FGF21 is produced from the liver during overfeeding and mitigates peripheral insulin resistance. DIO FGF21 liver-specific knockout, but not FGF21 adipose-specific knockout, mice have increased insulin resistance and decreased brown adipose tissue-mediated glucose disposal. These data are compatible with the concept that FGF21 functions physiologically as an insulin sensitizer under conditions of acute refeeding and overfeeding.

Brown adipose tissue regulates glucose homeostasis and insulin sensitivity.

Brown adipose tissue (BAT) is known to function in the dissipation of chemical energy in response to cold or excess feeding, and also has the capacity to modulate energy balance. To test the hypothesis that BAT is fundamental to the regulation of glucose homeostasis, we transplanted BAT from male donor mice into the visceral cavity of age- and sex-matched recipient mice. By 8-12 weeks following transplantation, recipient mice had improved glucose tolerance, increased insulin sensitivity, lower body weight, decreased fat mass, and a complete reversal of high-fat diet-induced insulin resistance. Increasing the quantity of BAT transplanted into recipient mice further improved the metabolic effects of transplantation. BAT transplantation increased insulin-stimulated glucose uptake in vivo into endogenous BAT, white adipose tissue (WAT), and heart muscle but, surprisingly, not skeletal muscle. The improved metabolic profile was lost when the BAT used for transplantation was obtained from Il6-knockout mice, demonstrating that BAT-derived IL-6 is required for the profound effects of BAT transplantation on glucose homeostasis and insulin sensitivity. These findings reveal a previously under-appreciated role for BAT in glucose metabolism.

Enhanced glycogen metabolism in adipose tissue decreases triglyceride mobilization.

Adipose tissue is a primary site for lipid storage containing trace amounts of glycogen. However, refeeding after a prolonged partial fast produces a marked transient spike in adipose glycogen, which dissipates in coordination with the initiation of lipid resynthesis. To further study the potential interplay between glycogen and lipid metabolism in adipose tissue, the aP2-PTG transgenic mouse line was utilized since it contains a 100- to 400-fold elevation of adipocyte glycogen levels that are mobilized upon fasting. To determine the fate of the released glucose 1-phosphate, a series of metabolic measurements were made. Basal and isoproterenol-stimulated lactate production in vitro was significantly increased in adipose tissue from transgenic animals. In parallel, basal and isoproterenol-induced release of nonesterified fatty acids (NEFAs) was significantly reduced in transgenic adipose tissue vs. control. Interestingly, glycerol release was unchanged between the genotypes, suggesting that enhanced triglyceride resynthesis was occurring in the transgenic tissue. Qualitatively similar results for NEFA and glycerol levels between wild-type and transgenic animals were obtained in vivo during fasting. Additionally, the physiological upregulation of the phosphoenolpyruvate carboxykinase cytosolic isoform (PEPCK-C) expression in adipose upon fasting was significantly blunted in transgenic mice. No changes in whole body metabolism were detected through indirect calorimetry. Yet weight loss following a weight gain/loss protocol was significantly impeded in the transgenic animals, indicating a further impairment in triglyceride mobilization. Cumulatively, these results support the notion that the adipocyte possesses a set point for glycogen, which is altered in response to nutritional cues, enabling the coordination of adipose glycogen turnover with lipid metabolism.

Stranger in a strange land: roles of glycogen turnover in adipose tissue metabolism.

Triglyceride storage in adipose tissue comprises the principal energy reserve in mammals. Additionally glucose can be stored as glycogen in the fed state, primarily in liver and skeletal muscle, for mobilization during times of energy deficit. Adipose tissue also contains glycogen stores albeit at very low levels. The physiological role of glycogen metabolism in adipocytes remains unclear. However, both classical literature and more recent work demonstrate that the dynamic regulation of adipose glycogen may serve as an energy sensing modality in the coordination of glucose and lipid metabolism in adipose tissue, especially during the fasted to fed transition.

Cross-talk between thyroid hormone receptor and liver X receptor regulatory pathways is revealed in a thyroid hormone resistance mouse model.

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor beta (TR-beta) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-beta knock-out mice, generalized expression of a non-ligand binding TR-beta protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7alpha-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-beta and liver X receptor alpha (LXR-alpha), which both bind to a direct repeat + 4 (DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-beta antagonized induction of this promoter by LXR-alpha. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-beta. A mechanism for cross-talk, and potential antagonism, between TR-beta and LXR-alpha is proposed.