Balancing gene transfection and cytotoxicity of nucleic acid carriers with focus on ocular and hepatic disorders: evaluation of hydrophobic and hydrophilic polyethyleneimine derivatives.

Polyethyleneimine (PEI) derivatives substituted by lactose, succinic acid or alkyl domains were evaluated as nonviral gene delivery vectors towards balancing gene transfection and cytotoxicity. The investigations were focused on pDNA transfection into arising retinal pigment epithelia (ARPE-19) and human hepatocellular carcinoma (HepG2) cell lines. The first mentioned cell line was chosen as motivated by the non-negligible number of ocular disorders linked to gene aberrations, whereas the second one is a cell line overexpressing the asialoglycoprotein receptor (ASGP-R), which can bind to galactose residues. The presence of short alkyl domains (C4 and C6), and particularly the succinylation of the PEI chains, improved the biological outputs of the gene vectors. The presence of hydrophobic units possibly enhances lytic activity, whereas the incorporation of succinic acid slightly reduces polymer-DNA interaction strength, thereby enabling more efficient intracellular unpacking and cargo release. Succinylation is also supposed to decrease cytotoxicity and avoid protein adsorption to the polyplexes. The presence of long carbon chains (for instance, C12) nevertheless, results in higher levels of cytotoxicity and respective lower transfection rates. The sugar-decorated polyplexes are overall less cytotoxic, but the presence of lactose moieties also leads to larger polyplexes and notably weak polymer-DNA binding, which compromise the transfection efficiency. Yet, along with the presence of short lytic alkyl domains, the double-substitution of PEI synergistically boosts gene transfection probably due to the uptake of higher DNA and polymer amounts without cell damage. Overall, the experimental data suggest that ocular and hepatic gene therapies may be potentialized by fine-tuning the hydrophobic-to-hydrophilic balance, and succinic acid is a favorable motif for the modification of PEI.

The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate.

WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.

Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.

While bile acids (BAs) have long been known to be essential in dietary lipid absorption and cholesterol catabolism, in recent years an important role for BAs as signalling molecules has emerged. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5 and activate nuclear hormone receptors such as farnesoid X receptor alpha (FXR-alpha; NR1H4). FXR-alpha regulates the enterohepatic recycling and biosynthesis of BAs by controlling the expression of genes such as the short heterodimer partner (SHP; NR0B2) that inhibits the activity of other nuclear receptors. The FXR-alpha-mediated SHP induction also underlies the downregulation of the hepatic fatty acid and triglyceride biosynthesis and very-low-density lipoprotein production mediated by sterol-regulatory-element-binding protein 1c. This indicates that BAs might be able to function beyond the control of BA homeostasis as general metabolic integrators. Here we show that the administration of BAs to mice increases energy expenditure in brown adipose tissue, preventing obesity and resistance to insulin. This novel metabolic effect of BAs is critically dependent on induction of the cyclic-AMP-dependent thyroid hormone activating enzyme type 2 iodothyronine deiodinase (D2) because it is lost in D2-/- mice. Treatment of brown adipocytes and human skeletal myocytes with BA increases D2 activity and oxygen consumption. These effects are independent of FXR-alpha, and instead are mediated by increased cAMP production that stems from the binding of BAs with the G-protein-coupled receptor TGR5. In both rodents and humans, the most thermogenically important tissues are specifically targeted by this mechanism because they coexpress D2 and TGR5. The BA-TGR5-cAMP-D2 signalling pathway is therefore a crucial mechanism for fine-tuning energy homeostasis that can be targeted to improve metabolic control.

Blockade of caspase cascade overcomes malaria-associated acute respiratory distress syndrome in mice.

Malaria is an enormous burden on global health that caused 409,000 deaths in 2019. Severe malaria can manifest in the lungs, an illness known as acute respiratory distress syndrome (ARDS). Not much is known about the development of malaria-associated ARDS (MA-ARDS), especially regarding cell death in the lungs. We had previously established a murine model that mimics various human ARDS aspects, such as pulmonary edema, hemorrhages, pleural effusion, and hypoxemia, using DBA/2 mice infected with Plasmodium berghei ANKA. Here, we explored the mechanisms and the involvement of apoptosis in this syndrome. We found that apoptosis contributes to the pathogenesis of MA-ARDS, primarily as facilitators of the alveolar-capillary barrier breakdown. The protection of pulmonary endothelium by inhibiting caspase activation could be a promising therapeutic strategy to prevent the pathogenicity of MA-ARDS. Therefore, intervention in the programmed death cell mechanism could help patients not to develop severe malaria.

The small polyphenolic molecule kaempferol increases cellular energy expenditure and thyroid hormone activation.

Disturbances in energy homeostasis can result in obesity and other metabolic diseases. Here we report a metabolic pathway present in normal human skeletal muscle myoblasts that is activated by the small polyphenolic molecule kaempferol (KPF). Treatment with KPF leads to an approximately 30% increase in skeletal myocyte oxygen consumption. The mechanism involves a several-fold increase in cyclic AMP (cAMP) generation and protein kinase A activation, and the effect of KPF can be mimicked via treatment with dibutyryl cAMP. Microarray and real-time PCR studies identified a set of metabolically relevant genes influenced by KPF including peroxisome proliferator-activated receptor gamma coactivator-1alpha, carnitine palmitoyl transferase-1, mitochondrial transcription factor 1, citrate synthase, and uncoupling protein-3, although KPF itself is not a direct mitochondrial uncoupler. The cAMP-responsive gene for type 2 iodothyronine deiodinase (D2), an intracellular enzyme that activates thyroid hormone (T3) for the nucleus, is approximately threefold upregulated by KPF; furthermore, the activity half-life for D2 is dramatically and selectively increased as well. The net effect is an approximately 10-fold stimulation of D2 activity as measured in cell sonicates, with a concurrent increase of approximately 2.6-fold in the rate of T3 production, which persists even 24 h after KPF has been removed from the system. The effects of KPF on D2 are independent of sirtuin activation and only weakly reproduced by other small polyphenolic molecules such as quercetin and fisetin. These data document a novel mechanism by which a xenobiotic-activated pathway can regulate metabolically important genes as well as thyroid hormone activation and thus may influence metabolic control in humans.

Expression patterns of WSB-1 and USP-33 underlie cell-specific posttranslational control of type 2 deiodinase in the rat brain.

The type 2 deiodinase (D2) activates thyroid hormone and constitutes an important source of 3,5,3',-triiodothyronine in the brain. D2 is inactivated via WSB-1 mediated ubiquitination but can be rescued from proteasomal degradation by USP-33 mediated deubiquitination. Using an in silico analysis of published array data, we found a significant positive correlation between the relative mRNA expression levels of WSB-1 and USP-33 in a set of 56 mouse tissues (r = 0.08; P < 0.04). Subsequently, we used in situ hybridization combined with immunocytochemistry in rat brain to show that in addition to neurons, WSB-1 and USP-33 are differently expressed in astrocytes and tanycytes, the two main D2 expressing cell types in this tissue. Tanycytes, which are thought to participate in the feedback regulation of TRH neurons express both WSB-1 and USP-33, indicating the potential for D2 ubiquitination and deubiquitination in these cells. Notably, only WSB-1 is expressed in glial fibrillary acidic protein-positive astrocytes throughout the brain. Although developmental and environmental signals are known to regulate the expression of WSB-1 and USP-33 in other tissues, our real-time PCR studies indicate that changes in thyroid status do not affect the expression of these genes in several rat brain regions, whereas in the mediobasal hypothalamus, changes in gene expression were minimal. In conclusion, the correlation between the relative mRNA levels of WSB-1 and USP-33 in numerous tissues that do not express D2 suggests that these ubiquitin-related enzymes share additional substrates besides D2. Furthermore, the data indicate that changes in WSB-1 and USP-33 expression are not part of the brain homeostatic response to hypothyroidism or hyperthyroidism.

Mice with impaired extrathyroidal thyroxine to 3,5,3'-triiodothyronine conversion maintain normal serum 3,5,3'-triiodothyronine concentrations.

For T(3) to mediate its biological effects, the prohormone T(4) must be activated by removal of an outer-ring iodine by the type 1 or 2 deiodinases (D1 and D2) with approximately 60% of the daily T(3) production in rodents being produced extrathyroidally through this pathway. To further define the role of these enzymes in thyroid hormone homeostasis, we backcrossed the targeted disruption of the Dio2 gene into C3H/HeJ (C3H) mice with genetically low D1 expression to create the C3H-D2KO mouse. Remarkably, these mice maintain euthyroid serum T(3) levels with normal growth and no decrease in expression of hepatic T(3)-responsive genes. However, serum T(4) is increased 1.2-fold relative to the already elevated C3H levels, and serum TSH is increased 1.4-fold. Despite these increases, thyroidal (125)I uptake indicates no difference in thyroidal activity between C3H-D2KO and C3H mice. Although C3H-D2KO hepatic and renal D1 activities were well below those observed in wild-type mice (approximately 0.1-fold for both), they were 8-fold and 2-fold higher, respectively, relative to C3H mice. Thyroidal D1 and cerebral cortical type 3 deiodinase activity were unchanged between C3H-D2KO and C3H mice. In conclusion, C3H-D2KO mice have notably elevated serum T(4) levels, and this, in conjunction with residual D1 activity, is likely an important role in the maintenance of euthyroid serum T(3) concentrations.

Is the FVB/N mouse strain truly resistant to diet-induced obesity?

C57Bl/6J mice are the gold standard animal model of diet-induced obesity. These animals become obese with higher adiposity, blood fasting glucose, triglycerides, and total cholesterol when fed a high-fat diet (HFD). Conversely, the FVB/N mouse line is thought to be resistant to diet-induced obesity, with low or no weight gain and adiposity in response to a HFD In this study, we investigated whether FVB/N mice are resistant or susceptible to metabolic disorder that is promoted by a HFD Biometric parameters and blood chemistry were analyzed in C57Bl/6J and FVB/N mice that were fed a chow diet or HFD Glucose and insulin sensitivity were assessed by performing the glucose tolerance test and measuring serum insulin/glucose and homeostasis model assessment-insulin resistance. Metabolism-related gene expression was investigated by real-time reverse transcription polymerase chain reaction. Adipocyte morphology and liver steatosis were evaluated using standard histology. FVB/N mice had higher adiposity than C57Bl/6J mice that were fed a chow diet and were glucose intolerant. FVB/N mice that were fed a HFD presented higher insulin resistance and greater liver steatosis. Epididymal white adipose tissue exhibited severe inflammation in FVB/N mice that were fed a HFD The FVB/N mouse strain is suitable for studies of diet-induced obesity, and the apparent lack of a HFD-induced response may reveal several strain-specific events that are triggered by a HFD Further studies of the FVB/N background may shed light on the complex multifactorial symptoms of obesity and metabolic syndrome.

A low-protein, high-carbohydrate diet increases browning in perirenal adipose tissue but not in inguinal adipose tissue.

OBJECTIVE: The aim of this study was to evaluate the browning and origin of fatty acids (FAs) in the maintenance of triacylglycerol (TG) storage and/or as fuel for thermogenesis in perirenal adipose tissue (periWAT) and inguinal adipose tissue (ingWAT) of rats fed a low-protein, high-carbohydrate (LPHC) diet. METHODS: LPHC (6% protein, 74% carbohydrate) or control (C; 17% protein, 63% carbohydrate) diets were administered to rats for 15 d. The tissues were stained with hematoxylin and eosin for histologic analysis. The content of uncoupling protein 1 (UCP1) was determined by immunofluorescence. Levels of T-box transcription factor (TBX1), PR domain containing 16 (PRDM16), adipose triacylglycerol lipase (ATGL), hormone-sensitive lipase, lipoprotein lipase (LPL), glycerokinase, phosphoenolpyruvate carboxykinase (PEPCK), glucose transporter 4, ß3-adrenergic receptor (AR), ß1-AR, protein kinase A (PKA), adenosine-monophosphate-activated protein kinase (AMPK), and phospho-AMPK were determined by immunoblotting. Serum fibroblast growth factor 21 (FGF21) was measured using a commercial kit (Student's t tests, P < 0.05). RESULTS: The LPHC diet increased FGF21 levels by 150-fold. The presence of multilocular adipocytes, combined with the increased contents of UCP1, TBX1, and PRDM16 in periWAT of LPHC-fed rats, suggested the occurrence of browning. The contents of ß1-AR and LPL were increased in the periWAT. The ingWAT showed higher ATGL and PEPCK levels, phospho-AMPK/AMPK ratio, and reduced ß3-AR and PKA levels. CONCLUSION: These findings suggest that browning occurred only in the periWAT and that higher utilization of FAs from blood lipoproteins acted as fuel for thermogenesis. Increased glycerol 3-phosphate generation by glyceroneogenesis increased FAs reesterification from lipolysis, explaining the increased TG storage in the ingWAT.

Differential effects of central leptin, insulin, or glucose administration during fasting on the hypothalamic-pituitary-thyroid axis and feeding-related neurons in the arcuate nucleus.

The reductions in circulating levels of leptin, insulin, and glucose with fasting serve as important homeostasis signals to neurons of the hypothalamic arcuate nucleus that synthesize neuropeptide Y (NPY)/agouti-related protein (AGRP) and alpha-MSH/cocaine and amphetamine-regulated transcript. Because the central administration of leptin is capable of preventing the inhibitory effects of fasting on TRH mRNA in hypophysiotropic neurons primarily through effects on the arcuate nucleus, we determined whether the continuous administration of 30 mU/d insulin or 648 microg/d glucose into the cerebrospinal fluid by osmotic minipump might also have similar effects on the hypothalamic-pituitary-thyroid axis. As anticipated, the intracerebroventricular infusion of leptin reduced fasting-induced elevations in NPY and AGRP mRNA and increased proopiomelanocortin and cocaine and amphetamine-regulated transcript mRNA in the arcuate nucleus. In addition, leptin prevented fasting-induced reduction in pro-TRH mRNA levels in the paraventricular nucleus and in circulating thyroid hormone levels. In contrast, whereas insulin increased proopiomelanocortin mRNA and both insulin and glucose reduced NPY mRNA in arcuate nucleus neurons, neither prevented the fasting-induced suppression in hypophysiotropic TRH mRNA or circulating thyroid hormone levels. We conclude that insulin and glucose only partially replicate the central effects of leptin and may not be essential components of the hypothalamic-pituitary-thyroid regulatory system during fasting.

Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism.

T(4), the main product of thyroid secretion, is a critical signal in plasma that mediates the TSH-negative feedback mechanism. As a prohormone, T(4) must be converted to T(3) to acquire biological activity; thus, type 2 iodothyronine deiodinase (D2) is expected to play a critical role in this feedback mechanism. However, the mechanistic details of this pathway are still missing because, counterintuitively, D2 activity is rapidly lost in the presence of T(4) by a ubiquitin-proteasomal mechanism. In the present study, we demonstrate that D2 and TSH are coexpressed in rat pituitary thyrotrophs and that hypothyroidism increases D2 expression in these cells. Studies using two murine-derived thyrotroph cells, TtT-97 and TalphaT1, demonstrate high expression of D2 in thyrotrophs and confirm its sensitivity to negative regulation by T(4)-induced proteasomal degradation of this enzyme. Despite this, expression of the Dio2 gene in TalphaT1 cells is higher than their T(4)-induced D2 ubiquitinating capacity. As a result, D2 activity and net T(3) production in these cells are sustained, even at free T(4) concentrations that are severalfold above the physiological range. In this system, free T(4) concentrations and net D2-mediated T(3) production correlated negatively with TSHbeta gene expression. These results resolve the apparent paradox between the homeostatic regulation of D2 and its role in mediating the critical mechanism by which T(4) triggers the TSH-negative feedback.

Type 2 iodothyronine selenodeiodinase is expressed throughout the mouse skeleton and in the MC3T3-E1 mouse osteoblastic cell line during differentiation.

Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nM, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5-7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30-40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNFalpha, TGFbeta, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.

Type 1 iodothyronine deiodinase is a sensitive marker of peripheral thyroid status in the mouse.

Mice with one thyroid hormone receptor (TR) alpha-1 allele encoding a dominant negative mutant receptor (TR alpha1(PV/+)) have persistently elevated serum T3 levels (1.9-fold above normal). They also have markedly increased hepatic type 1 iodothyronine deiodinase (D1) mRNA and enzyme activity (4- to 5-fold), whereas other hepatic T3-responsive genes, such as Spot14 and mitochondrial alpha-glycerol phosphate dehydrogenase (alpha-GPD), are only 0.7-fold and 1.7-fold that of wild-type littermates (TR alpha1+/+). To determine the cause of the disproportionate elevation of D1, TR alpha1+/+ and TR alpha1(PV/+) mice were rendered hypothyroid and then treated with T3. Hypothyroidism decreased hepatic D1, Spot14, and alpha-GPD mRNA to similar levels in TR alpha1+/+ and TR alpha1(PV/+) mice, whereas T3 administration caused an approximately 175-fold elevation of D1 mRNA but only a 3- to 6-fold increases in Spot14 and alpha-GPD mRNAs. Interestingly, the hypothyroidism-induced increase in cerebrocortical type 2 iodothyronine deiodinase activity was 3 times greater in the TR alpha1(PV/+) mice, and these mice had no T3-dependent induction of type 3 iodothyronine deiodinase. Thus, the marked responsiveness of hepatic D1 to T3 relative to other genes, such as Spot14 and alpha-GPD, explains the relatively large effect of the modest increase in serum T3 in the TR alpha1(PV/+) mice, and TR alpha plays a key role in T3-dependent positive and negative regulation of the deiodinases in the cerebral cortex.

Mice with targeted disruption of the Dio2 gene have cold-induced overexpression of the uncoupling protein 1 gene but fail to increase brown adipose tissue lipogenesis and adaptive thermogenesis.

The Dio2 gene encodes the type 2 deiodinase (D2) that activates thyroxine (T4) to 3,3',5-triiodothyronine (T3), the disruption of which (Dio2(-/-)) results in brown adipose tissue (BAT)-specific hypothyroidism in an otherwise euthyroid animal. In the present studies, cold exposure increased Dio2(-/-) BAT sympathetic stimulation approximately 10-fold (normal approximately 4-fold); as a result, lipolysis, as well as the mRNA levels of uncoupling protein 1, guanosine monophosphate reductase, and peroxisome proliferator-activated receptor gamma coactivator 1, increased well above the levels detected in the cold-exposed wild-type animals. The sustained Dio2(-/-) BAT adrenergic hyperresponse suppressed the three- to fourfold stimulation of BAT lipogenesis normally seen after 24-48 h in the cold. Pharmacological suppression of lipogenesis with betabeta'-methyl-substituted alpha-omega-dicarboxylic acids of C14-C18 in wild-type animals also impaired adaptive thermogenesis in the BAT. These data constitute the first evidence that reduced adrenergic responsiveness does not limit cold-induced adaptive thermogenesis. Instead, the resulting compensatory hyperadrenergic stimulation prevents the otherwise normal stimulation in BAT lipogenesis during cold exposure, rapidly exhausting the availability of fatty acids. The latter is the preponderant determinant of the impaired adaptive thermogenesis and hypothermia in cold-exposed Dio2(-/-) mice.

Bacterial lipopolysaccharide (LPS)-induced type 2 iodothyronine deiodinase (D2) activation in the mediobasal hypothalamus (MBH) is independent of the LPS-induced fall in serum thyroid hormone levels.

By administration of bacterial lipopolysaccharide (LPS) to intact and T4-replaced thyroidectomized rats, we demonstrate that in contrast to the cortex and anterior pituitary, there is a persistent increase in type 2 iodothyronine deiodinase (D2) activity in the mediobasal hypothalamus (MBH). We propose that endotoxin-induced D2 activation in the MBH is independent of circulating levels of thyroid hormone and that this mechanism may contribute to central hypothyroidism associated with infection.

Adaptive activation of thyroid hormone and energy expenditure.

The mechanisms by which thyroid hormone accelerates energy expenditure are poorly understood. In the brown adipose tissue (BAT), activation of thyroid hormone by type 2 iodothyronine deiodinase (D2) has been known to play a role in adaptive energy expenditure during cold exposure in human newborns and other small mammals. Although BAT is not present in significant amounts in normal adult humans, recent studies have found substantial amounts of D2 in skeletal muscle, a metabolically relevant tissue in humans. This article reviews current biological knowledge about D2 and adaptive T3 production and their roles in energy expenditure.

Inactivation of the adrenergic receptor ß2 disrupts glucose homeostasis in mice.

Three types of beta adrenergic receptors (ARß1-3) mediate the sympathetic activation of brown adipose tissue (BAT), the key thermogenic site for mice which is also present in adult humans. In this study, we evaluated adaptive thermogenesis and metabolic profile of a mouse with Arß2 knockout (ARß2KO). At room temperature, ARß2KO mice have normal core temperature and, upon acute cold exposure (4 °C for 4 h), ARß2KO mice accelerate energy expenditure normally and attempt to maintain body temperature. ARß2KO mice also exhibited normal interscapular BAT thermal profiles during a 30-min infusion of norepinephrine or dobutamine, possibly due to marked elevation of interscapular BAT (iBAT) and of Arß1, and Arß3 mRNA levels. In addition, ARß2KO mice exhibit similar body weight, adiposity, fasting plasma glucose, cholesterol, and triglycerides when compared with WT controls, but exhibit marked fasting hyperinsulinemia and elevation in hepatic Pepck (Pck1) mRNA levels. The animals were fed a high-fat diet (40% fat) for 6 weeks, ARß2KO mice doubled their caloric intake, accelerated energy expenditure, and induced Ucp1 expression in a manner similar to WT controls, exhibiting a similar body weight gain and increase in the size of white adipocytes to the WT controls. However, ARß2KO mice maintain fasting hyperglycemia as compared with WT controls despite very elevated insulin levels, but similar degrees of liver steatosis and hyperlipidemia. In conclusion, inactivation of the ARß2KO pathway preserves cold- and diet-induced adaptive thermogenesis but disrupts glucose homeostasis possibly by accelerating hepatic glucose production and insulin secretion. Feeding on a high-fat diet worsens the metabolic imbalance, with significant fasting hyperglycemia but similar liver structure and lipid profile to the WT controls.

ß(1) Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice.

Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple ß-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the ß(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the ß(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the ß(1) gene (KO of ß(1) adrenergic receptor (ß(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, ß(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, ß(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the ß(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis.

Regulation of thyroid hormone activation via the liver X-receptor/retinoid X-receptor pathway.

Thyroid hormone receptor (TR) and liver X-receptor (LXR) are the master regulators of lipid metabolism. Remarkably, a mouse with a targeted deletion of both LXR alpha and LXR beta is resistant to western diet-induced obesity, and exhibits ectopic liver expression of the thyroid hormone activating type 2 deiodinase (D2). We hypothesized that LXR/retinoid X-receptor (RXR) signaling inhibits hepatic D2 expression, and studied this using a luciferase reporter containing the human DIO2 (hDIO2) promoter in HepG2 cells. Given that, in contrast to mammals, the chicken liver normally expresses D2, the chicken DIO2 (cDIO2) promoter was also studied. 22(R)-OH-cholesterol negatively regulated hDIO2 in a dose-dependent manner (100 microM, approximately twofold), while it failed to affect the cDIO2 promoter. Truncations in the hDIO2 promoter identified the region -901 to -584 bp as critical for negative regulation. We also investigated if 9-cis retinoic acid (9-cis RA), the ligand for the heterodimeric partner of TR and LXR, RXR, could regulate the hDIO2 promoter. Notably, 9-cis RA repressed the hDIO2 luciferase reporter (1 microM, approximately fourfold) in a dose-dependent manner, while coexpression of an inactive mutant RXR abolished this effect. However, it is unlikely that RXR homodimers mediate the repression of hDIO2 since mutagenesis of a DR-1 at -506 bp did not interfere with 9-cis RA-mediated repression. Our data indicate that hDIO2 transcription is negatively regulated by both 22(R)-OH-cholesterol and 9-cis RA, which is consistent with LXR/RXR involvement. In vivo, the inhibition of D2-mediated tri-iodothyronine (T(3)) production by cholesterol/9-cis RA could function as a feedback loop, given that T(3) decreases hepatic cholesterol levels.

Absence of thyroid hormone activation during development underlies a permanent defect in adaptive thermogenesis.

Type 2 deiodinase (D2), which is highly expressed in brown adipose tissue (BAT), is an enzyme that amplifies thyroid hormone signaling in individual cells. Mice with inactivation of the D2 pathway (D2KO) exhibit dramatically impaired thermogenesis in BAT, leading to hypothermia during cold exposure and a greater susceptibility to diet-induced obesity. This was interpreted as a result of defective acute activation of BAT D2. Here we report that the adult D2KO BAT has a permanent thermogenic defect that stems from impaired embryonic BAT development. D2KO embryos have normal serum T3 but due to lack of D2-generated T3 in BAT, this tissue exhibits decreased expression of genes defining BAT identity [i.e. UCP1, PGC-1alpha and Dio2 (nonfunctional)], which results in impaired differentiation and oxidative capacity. Coinciding with a reduction of these T3-responsive genes, there is oxidative stress that in a cell model of brown adipogenesis can be linked to decreased insulin signaling and decreased adipogenesis. This discovery highlights the importance of deiodinase-controlled thyroid hormone signaling in BAT development, where it has important metabolic repercussions for energy homeostasis in adulthood.