Inactivation of UCP1 and the glycerol phosphate cycle synergistically increases energy expenditure to resist diet-induced obesity

Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 degrees C, provided prior adaptation occurred at 20 degrees C. Obesity resistant Gdm(-/-).Ucp1(-/-) mice maintained at 28 degrees C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency.     

Gab2 deficiency suppresses high-fat diet-induced obesity by reducing adipose tissue inflammation and increasing brown adipose function in mice

Obesity is caused by a long-term imbalance between energy intake and consumption and is regulated by multiple signals. This study investigated the effect of signaling scaffolding protein Gab2 on obesity and its relevant regulation mechanism. Gab2 knockout (KO) and wild-type (WT) mice were fed with a standard diet (SD) or high-fat diet (HFD) for 12 weeks. The results showed that the a high-fat diet-induced Gab2 expression in adipose tissues, but deletion of Gab2 attenuated weight gain and improved glucose tolerance in mice fed with a high-fat diet. White adipose tissue and systemic inflammations were reduced in HFD-fed Gab2 deficiency mice. Gab2 deficiency increased the expression of Ucp1 and other thermogenic genes in brown adipose tissue. Furthermore, the regulation of Gab2 on the mature differentiation and function of adipocytes was investigated in vitro using primary or immortalized brown preadipocytes. The expression of brown fat-selective genes was found to be elevated in differentiated adipocytes without Gab2. The mechanism of Gab2 regulating Ucp1 expression in brown adipocytes involved with its downstream PI3K (p85)-Akt-FoxO1 signaling pathway. Our research suggests that deletion of Gab2 suppresses diet-induced obesity by multiple pathways and Gab2 may be a novel therapeutic target for the treatment of obesity and associated complications.Subject terms: Fat metabolism, Obesity     

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2–4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.     

UCP1 deficiency increases susceptibility to diet-induced obesity with age

Loss of nonshivering thermogenesis in mice by inactivation of the mitochondrial uncoupling protein gene (Ucp1-/- mice) causes increased sensitivity to cold and unexpected resistance to diet-induced obesity at a young age. To clarify the role of UCP1 in body weight regulation throughout life and influence of UCP1 deficiency on longevity, we longitudinally analyzed the phenotypes of Ucp1-/- mice maintained in a room at 23 degrees C. There was no difference in body weight and lifespan between genotypes under the standard chow diet condition, whereas the mutant mice developed obesity with age under the high-fat (HF) diet condition. Compared with Ucp1+/+ mice, Ucp1-/- mice showed increased expression of genes related to thermogenesis and fatty acid metabolism, such as beta3-adrenergic receptor, in adipose tissues of the 3-month-old mutants; however, the augmented expression was reduced in Ucp1+/+ mice in 11-month-old Ucp1-/- mice fed the HF diet. Likewise, the increased levels of UCP3 and cAMP-dependent protein kinase in the brown adipose tissue of Ucp1-/- mice given the standard diet were decreased significantly in that of Ucp1-/- mice fed the HF diet, which animals showed impaired norepinephrine-induced lipolysis in their adipose tissues. These results suggest profound attenuation of beta-adrenergic responsiveness and fatty acid utilization in Ucp1-/- mice fed the HF diet, bringing them to late-onset obesity. Our findings provide evidence that UCP1 is neither essential for body weight regulation nor for longevity under conditions of standard diet and normal housing temperature, but deficiency increases susceptibility to obesity with age in combination with HF diet.     

Raspberry promotes brown and beige adipocyte development in mice fed high-fat diet through activation of AMP-activated protein kinase (AMPK) α1

Development of brown and beige/brite adipocytes increases thermogenesis and helps to reduce obesity and metabolic syndrome. Our previous study suggests that dietary raspberry can ameliorate metabolic syndromes in diet-induced obese mice. Here, we further evaluated the effects of raspberry on energy expenditure and adaptive thermogenesis and determined whether these effects were mediated by AMP-activated protein kinase (AMPK). Mice deficient in the catalytic subunit of AMPKα1 and wild-type (WT) mice were fed a high-fat diet (HFD) or HFD supplemented with 5% raspberry (RAS) for 10 weeks. The thermogenic program and related regulatory factors in adipose tissue were assessed. RAS improved the insulin sensitivity and reduced fat mass in WT mice but not in AMPKα1^-/- mice. In the absence of AMPKα1, RAS failed to increase oxygen consumption and heat production. Consistent with this, the thermogenic gene expression in brown adipose tissue and brown-like adipocyte formation in subcutaneous adipose tissue were not induced by RAS in AMPKα1^-/- mice. In conclusion, AMPKα1 is indispensable for the effects of RAS on brown and beige/brite adipocyte development, and prevention of obesity and metabolic dysfunction.     

Uncoupling protein 1 expression and high-fat diets

Uncoupling protein 1 (Ucp1) is the key component of β-adrenergically controlled nonshivering thermogenesis in brown adipocytes. This process combusts stored and nutrient energy as heat. Cold exposure not only activates Ucp1-mediated thermogenesis to maintain normothermia but also results in adaptive thermogenesis, i.e., the recruitment of thermogenic capacity in brown adipose tissue. As a hallmark of adaptive thermogenesis, Ucp1 synthesis is increased proportionally to temperature and duration of exposure. Beyond this classical thermoregulatory function, it has been suggested that Ucp1-mediated thermogenesis can also be employed for metabolic thermogenesis to prevent the development of obesity. Accordingly, in times of excess caloric intake, one may expect a positive regulation of Ucp1. The general impression from an overview of the present literature is, indeed, an increased brown adipose tissue Ucp1 mRNA and protein content after feeding a high-fat diet (HFD) to mice and rats. The reported increases are very variable in magnitude, and the effect size seems to be independent of dietary fat content and duration of the feeding trial. In white adipose tissue depots Ucp1 mRNA is generally downregulated by HFD, indicating a decline in the number of interspersed brown adipocytes.     

Ablation of NG2 proteoglycan leads to deficits in brown fat function and to adult onset obesity

Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O(2) consumption and CO(2) production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes.     

Inherent plasticity of brown adipogenesis in white fat of mice allows for recovery from effects of post-natal malnutrition

Interscapular brown adipose tissue (iBAT) is formed during fetal development and stable for the life span of the mouse. In addition, brown adipocytes also appear in white fat depots (wBAT) between 10 and 21 days of age in mice maintained at a room temperature of 23 °C. However, this expression is transient. By 60 days of age the brown adipocytes have disappeared, but they can re-emerge if the adult mouse is exposed to the cold (5 °C) or treated with β3-adrenergic agonists. Since the number of brown adipocytes that can be induced in white fat influences the capacity of the mouse to resist the obese state, we determined the effects of the nutritional conditions on post-natal development (birth to 21 days) of wBAT and its long-term effects on diet-induced obesity (DIO). Under-nutrition caused essentially complete suppression of wBAT in inguinal fat at 21 days of age, as indicated by expression of Ucp1 and genes of mitochondrial structure and function based upon microarray and qRT-PCR analysis, whereas over-nutrition had no discernible effects on wBAT induction. Surprisingly, the suppression of wBAT at 21 days of age did not affect DIO in adult mice maintained at 23 °C, nor did it affect the reduction in obesity or cold tolerance when DIO mice were exposed to the cold at 5 °C for one week. Gene expression analysis indicated that mice raised under conditions that suppressed wBAT at 21 days of age were able to normally induce wBAT as adults. Therefore, neither severe hypoleptinemia nor hypoinsulinemia during suckling permanently impaired brown adipogenesis in white fat. In addition, energy balance studies of DIO mice exposed to cold indicates that mice with reduced adipose stores preferentially increased food intake, whereas those with larger adipose tissue depots preferred to utilize energy from their adipose stores.     

Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity

Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity.     

Deficiency of PTP1B in POMC neurons leads to alterations in energy balance and homeostatic response to cold exposure

The adipose tissue-derived hormone leptin regulates energy balance through catabolic effects on central circuits, including proopiomelanocortin (POMC) neurons. Leptin activation of POMC neurons increases thermogenesis and locomotor activity. Protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of leptin signaling. POMC neuron-specific deletion of PTP1B in mice results in reduced high-fat diet-induced body weight and adiposity gain due to increased energy expenditure and greater leptin sensitivity. Mice lacking the leptin gene (ob/ob mice) are hypothermic and cold intolerant, whereas leptin delivery to ob/ob mice induces thermogenesis via increased sympathetic activity to brown adipose tissue (BAT). Here, we examined whether POMC PTP1B mediates the thermoregulatory response of CNS leptin signaling by evaluating food intake, body weight, core temperature (T(C)), and spontaneous physical activity (SPA) in response to either exogenous leptin or 4-day cold exposure (4°C) in male POMC-Ptp1b-deficient mice compared with wild-type controls. POMC-Ptp1b(-/-) mice were hypersensitive to leptin-induced food intake and body weight suppression compared with wild types, yet they displayed similar leptin-induced increases in T(C). Interestingly, POMC-Ptp1b(-/-) mice had increased BAT weight and elevated plasma triiodothyronine (T(3)) levels in response to a 4-day cold challenge, as well as reduced SPA 24 h after cold exposure, relative to controls. These data show that PTP1B in POMC neurons plays a role in short-term cold-induced reduction of SPA and may influence cold-induced thermogenesis via enhanced activation of the thyroid axis.     

Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4^−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4^−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4^−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4^−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4^−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.     

The Chemical Uncoupler 2,4-Dinitrophenol (DNP) Protects against Diet-induced Obesity and Improves Energy Homeostasis in Mice at Thermoneutrality

The chemical uncoupler 2,4-dinitrophenol (DNP) was an effective and widely used weight loss drug in the early 1930s. However, the physiology of DNP has not been studied in detail because toxicity, including hyperthermia and death, reduced interest in the clinical use of chemical uncouplers. To investigate DNP action, mice fed a high fat diet and housed at 30 °C (to minimize facultative thermogenesis) were treated with 800 mg/liter DNP in drinking water. DNP treatment increased energy expenditure by ∼17%, but did not change food intake. DNP-treated mice weighed 26% less than controls after 2 months of treatment due to decreased fat mass, without a change in lean mass. DNP improved glucose tolerance and reduced hepatic steatosis without observed toxicity. DNP treatment also reduced circulating T3 and T4 levels, Ucp1 expression, and brown adipose tissue activity, demonstrating that DNP-mediated heat generation substituted for brown adipose tissue thermogenesis. At 22 °C, a typical vivarium temperature that is below thermoneutrality, DNP treatment had no effect on body weight, adiposity, or glucose homeostasis. Thus, environmental temperature should be considered when assessing an anti-obesity drug in mice, particularly agents acting on energy expenditure. Furthermore, the beneficial effects of DNP suggest that chemical uncouplers deserve further investigation for the treatment of obesity and its comorbidities.     

MicroRNA 34a Inhibits Beige and Brown Fat Formation in Obesity in Part by Suppressing Adipocyte Fibroblast Growth Factor 21 Signaling and SIRT1 Function

Brown fat generates heat through uncoupled respiration, protecting against hypothermia and obesity. Adult humans have brown fat, but the amounts and activities are substantially decreased in obesity, by unknown mechanisms. Here we show that elevated microRNA 34a (miR-34a) in obesity inhibits fat browning in part by suppressing the browning activators fibroblast growth factor 21 (FGF21) and SIRT1. Lentivirus-mediated downregulation of miR-34a in mice with diet-induced obesity reduced adiposity, improved serum profiles, increased the mitochondrial DNA copy number, and increased oxidative function in adipose tissue in both BALB/c and C57BL/6 mice. Remarkably, downregulation of miR-34a increased coexpression of the beige fat-specific marker CD137 and the browning marker UCP1 in all types of white fat, including visceral fat, and promoted additional browning in brown fat. Mechanistically, downregulation of miR-34a increased expression of the FGF21 receptor components, FGFR1 and βKL, and also that of SIRT1, resulting in FGF21/SIRT1-dependent deacetylation of PGC-1α and induction of the browning genes Ucp1, Pgc-1α, and Prdm16. Importantly, anti-miR-34a-mediated beneficial effects, including decreased adiposity, are likely from multiple tissues, since downregulation of miR-34a also improves hepatic FGF21 signaling and lipid oxidation. This study identifies miR-34a as an inhibitor of beige and brown fat formation, providing a potential target for treating obesity-related diseases.     

Reduced lipogenesis in cafeteria-fed rats exhibiting diet-induced thermogenesis

Fatty-acid synthesis has been measured in vivo with³H₂O in cafeteria-fed rats exhibiting diet-induced thermogenesis. Synthesis was decreased in brown adipose tissue, the liver, white adipose tissue, and the carcass of the cafeteria-fed animals compared to rats fed the normal stock diet. Whole-body synthesis was also decreased in the cafeteria-fed group. Diet-induced thermogenesis, in contrast to cold-induced non-shivering thermogenesis does not lead to increased fatty-acid synthesis and this is presumably due to the inhibitory effects on lipogenesis of the high dietary fat intake characteristic of cafeteria diets. The results also indicate that the energy cost of body fat deposition in cafeteria-fed rats is lower than in animals fed a low-fat/high-carbohydrate stock diet.     

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.     

Mfn2 deletion in brown adipose tissue protects from insulin resistance and impairs thermogenesis

BAT‐controlled thermogenic activity is thought to be required for its capacity to prevent the development of insulin resistance. This hypothesis predicts that mediators of thermogenesis may help prevent diet‐induced insulin resistance. We report that the mitochondrial fusion protein Mitofusin 2 (Mfn2) in BAT is essential for cold‐stimulated thermogenesis, but promotes insulin resistance in obese mice. Mfn2 deletion in mice through Ucp1‐cre (BAT‐Mfn2‐KO) causes BAT lipohypertrophy and cold intolerance. Surprisingly however, deletion of Mfn2 in mice fed a high fat diet (HFD) results in improved insulin sensitivity and resistance to obesity, while impaired cold‐stimulated thermogenesis is maintained. Improvement in insulin sensitivity is associated with a gender‐specific remodeling of BAT mitochondrial function. In females, BAT mitochondria increase their efficiency for ATP‐synthesizing fat oxidation, whereas in BAT from males, complex I‐driven respiration is decreased and glycolytic capacity is increased. Thus, BAT adaptation to obesity is regulated by Mfn2 and with BAT‐Mfn2 absent, BAT contribution to prevention of insulin resistance is independent and inversely correlated to whole‐body cold‐stimulated thermogenesis.     

Apple polyphenols induce browning of white adipose tissue

We and others have shown that apple polyphenols decrease adipose tissue mass. To better understand the underlying mechanisms and to expand clinical applicability, we herein examine whether apple polyphenols induce adipose thermogenic adaptations (browning) and prevent diet-induced obesity and related insulin resistance. In mice fed a standard diet, daily apple polyphenol consumption induced thermogenic adaptations in inguinal white adipose tissue (iWAT), based on increases in the expression of brown/beige adipocyte selective genes (Ucp1, Cidea, Tbx1, Cd137) and protein content of uncoupling protein 1 and mitochondrial oxidative phosphorylation enzymes. Among the upstream regulatory factors of browning, fibroblast growth factor 21 (FGF21) and peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC-1α) levels were concomitantly up-regulated by apple polyphenols. In the primary cell culture experiment, the results did not support a direct action of apple polyphenols on beige adipogenesis. Instead, apple polyphenols increased tyrosine hydroxylase (a rate-limiting enzyme of catecholamine synthesis) in iWAT, which activates the adipocyte thermogenic program possibly via intratissue cellular communications. In high-fat fed mice, apple polyphenols induced beige adipocyte development in iWAT, reduced fat accumulation, and increased glucose disposal rates in the glucose and insulin tolerance tests. Taken together, dietary administration of apple polyphenols induced beige adipocyte development in iWAT possibly via activation/induction of the peripheral catecholamine synthesis–FGF21–PGC-1α cascade. Results from diet-induced obese mice indicate that apple polyphenols have therapeutic potential for obesity and related metabolic disorders.     

Lipocalin 2 Regulates Brown Fat Activation via a Nonadrenergic Activation Mechanism

In this study, we report that lipocalin 2 (Lcn2), a recently characterized adipokine/cytokine, is a novel regulator of brown adipose tissue (BAT) activation by modulating the adrenergic independent p38 MAPK-PGC-1α-UCP1 pathway. Global Lcn2 knock-out (Lcn2^−/−) mice have defective BAT thermogenic activation caused by cold stimulation and decreased BAT activity under high fat diet-induced obesity. Nevertheless, Lcn2^−/− mice maintain normal sympathetic nervous system activation as evidenced by normal catecholamine release and lipolytic activity in response to cold stimulation. Further studies showed that Lcn2 deficiency impairs peroxisomal and mitochondrial oxidation of lipids and attenuates cold-induced Pgc1a and Ucp1 expression and p38 MAPK phosphorylation in BAT. Moreover, in vitro studies showed that Lcn2 deficiency reduces the thermogenic activity of brown adipocytes. Lcn2^−/− differentiated brown adipocytes have significantly decreased expression levels of brown fat markers, decreased p38 MAPK phosphorylation, and decreased mitochondrial oxidation capacity. However, Lcn2^−/− brown adipocytes have normal norepinephrine-stimulated p38 MAPK and hormone-sensitive lipase phosphorylation and Pgc1a and Ucp1 expression, suggesting an intact β-adrenergic signaling activation. More intriguingly, recombinant Lcn2 was able to significantly stimulate p38 MAPK phosphorylation in brown adipocytes. Activating peroxisome proliferator-activated receptor γ, a downstream effector of PGC-1α, by thiazolidinedione administration fully reverses the BAT function of Lcn2^−/− mice. Our findings provide evidence for the novel role Lcn2 plays in oxidative metabolism and BAT activation via an adrenergic independent mechanism.