Skeletal muscle-secreted DLPC orchestrates systemic energy homeostasis by enhancing adipose browning

Obesity is the number one cardiovascular risk factor for both men and women and is a complex condition. Although a sex dimorphism on vascular function has already been noted, the underlying processes remain unclear. The Rho-kinase pathway has a unique role in controlling vascular tone, and in obese male mice, hyperactivation of this system results in worsened vascular constriction. We investigated whether female mice exhibit decreased Rho-kinase activation as a protective mechanism in obesity. We exposed male and female mice to a high-fat diet (HFD) for 14 weeks. At the end, energy expenditure, glucose tolerance, adipose tissue inflammation, and vascular function were investigated. Male mice were more sensitive to HFD-induced body weight gain, glucose tolerance, and inflammation than female mice. After establishing obesity, female mice demonstrated increase in energy expenditure, characterized by an increase in heat, whereas male mice did not. Interestingly, obese female mice, but not male, displayed attenuated vascular contractility to different agonists, such difference was blunted by inhibition of Rho-kinase, which was accompanied by a suppressed Rho-kinase activation, measured by Western blot. Finally, aortae from obese male mice displayed an exacerbated inflammation, whereas obese female demonstrated a mild vascular inflammation. In obesity, female mice demonstrate a vascular protective mechanism-suppression of vascular Rho-kinase-to minimize the cardiovascular risk associated with obesity, whereas male mice do not generate any adaptive response. Future investigations can help to understand how Rho-kinase becomes suppressed in female during obesity.

Introduction: Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is a major biologically active natriuretic peptide (NP) receptor that synthesizes intracellular second-messenger cGMP in response to hormone binding. Atrial and brain natriuretic peptides (ANP and BNP) are the major cardiac peptide hormones responsible for fluid and electrolytes homeostasis, vasorelaxant, antiproliferative, and antihypertrophic responses, which lead to a reduction in blood pressure (BP) and cardiac dysfunction. Methods: Generation of CM-specific Npr1 deleted mice, a floxed Npr1 mouse line was generated with genotype of confirmed Npr1Flox/Flox ( f/f ) alleles. The CM-specific Cre mice having αMHC/Mer- Cre trans allele (Tamoxifen-inducible; Jackson Laboratory) were mated with WT f/f mice to generate F1 heterozygous CM- Cre;Npr1f/+ mice. The F1 hybrids having both Cre and Flox alleles were mated with original wild-type (WT) f/f mice to produce conditional homozygous null CM- Cre;Npr1f/- (CM-KO) mice. Consecutive administration of Tamoxifen (0.1 mg/g/day) for 5 days to adult (6-8 wks) male and female CM- Cre; Npr1f/- mice achieved inducible CM-specific inactivation of Npr1 in cardiomyocytes. The heart structure and function were determined by echocardiography. Results: The results showed that cardiac hypertrophy, left ventricular end-systolic and diastolic dimension (LVED-s and LVED-d), and posterior wall thickness (PWT) were significantly increased in male KO mice (4.4 + 0.1, 3.2 + 0.1; p<0.05) than female KO mice (4.1 + 0.1, 3.2 + 0.2; p<0.05) compared with WT male mice (3.7 + 0.1, 2.3 + 0.1; p<0.05) and female mice (4.2 + 0.1, 3.1 + 0.1; p<0.05). PWT was also significantly increased in both male and female KO mice compared with WT mice. Heart wight/body weight (HW/BW) ratio was significantly (p<0.005) increased in male KO mice than female KO mice compared with WT mice. The fractional shortening was significantly (p<0.05) compromised with greater magnitude in male KO mice than female KO mice. The f/f mice presented a more preserved contractility in the LV wall, we found that CM-KO mice showed a decreased in contractility in both sexes and this decrease was found in all parts of the LV, suggesting a weakness in the cardiomyocytes of CM-KO mice. Conclusion: The results suggest that deletion of Npr1 in cardiomyocytes provokes cardiac hypertrophy and dysfunction with remodeling in both sexes with higher magnitudes in males than females. Supported by NIH/NIDDK grant (DK133833).

Effects of selenium supplementation on diet-induced obesity in mice with a disruption of the selenocysteine lyase gene

Objective: We recently demonstrated that male, but not female mice fed a high fat (HF) diet developed obesity-hypertension. Castration of female mice conferred obesity-hypertension. The hypothesis of this study is estrogen administration reduces obesity-hypertension in intact male and ovariectomized female HF-fed mice. Methods and Results: Male (8 weeks old) and ovariectomized female (10 weeks old) C57BL/6 mice were fed a HF diet (60% kcal as fat) for 16 weeks. Exogenous estrogen (36g/ml 17-estradiol) or vehicle (sesame oil) were continuously delivered via silastic tubes implanted subcutaneously in HF-fed male mice from the beginning of HF feeding. For females, HF diet and estrogen administration began 2 weeks after ovariectomy. There was no effect of estrogen administration on the development of obesity in male mice. However, female mice administered estrogen showed a marked reduction in the development of obesity (Body weight: Vehicle: 48.7 ± 1.7 g; Estrogen: 38.9 ± 1.9 g; p<0.05). Administration of estrogen to male HF-fed mice improved glucose tolerance (AUC; estrogen: 42150 ± 1865; Vehicle: 48463 ± 1899; p =0.025). Systolic blood pressure (SBP) was not influenced by estrogen administration in male mice (SBP: Vehicle: 147 ± 4 mmHg; Estrogen: 147 ± 3 mmHg). However, estrogen decreased SBP in female HF-fed ovariectomized mice (SBP: Vehicle: 153 ± 11 mmHg; Estrogen: 137 ± 5 mmHg; p=0.019). Conclusion: Administration of estrogen to male obese mice had no effect on blood pressure, but improved glucose homeostasis. In ovariectomized HF-fed females, estrogen administration reduced blood pressure. These results suggest that resistance to obesity-hypertension in females may be estrogen-mediated.

<p dir="ltr">While leptin control of metabolism is primarily viewed as centrally mediated, leptin has also been shown to directly regulate adipocyte function. However, the impact of the peripheral effects of leptin on systemic metabolism, especially in the context of obesity, remains unclear. To address this question, we selectively restored adipocyte leptin receptor (LEPR) expression in obese male and female LEPR conditional KO mice. Adipocyte LEPR restoration did not affect body weight but selectively increased brown adipose tissue (BAT) mass in male mice. This was associated with increased energy expenditure, smaller BAT adipocytes, lower triglycerides content, and increased markers of browning and lipolysis exclusively in males. Additionally, adipocyte LEPR restoration enhanced the expression of markers of endothelial cell and angiogenesis in male BAT, supporting increased local vascularization. Improved BAT function in males was also associated with lower HbA1c, better insulin sensitivity, reduced systolic blood pressure, decreased arterial stiffness and improved endothelial function. Lastly, adipocyte LEPR restoration lowered circulating pro-inflammatory cytokines and reduced tissue inflammation in the aorta and the heart, again in males only. These findings reveal a critical role for adipocyte leptin signaling in regulating BAT function and emphasize its importance in maintaining glycemic and cardiovascular health in males with obesity.</p>

Obese male mice (ob/ob) are normally sensitive to the satiating effect of CCK-8

Overnutrition engenders the expansion of adipose tissue and the accumulation of immune cells, in particular, macrophages, in the adipose tissue, leading to chronic low-grade inflammation and insulin resistance. In obesity, several proinflammatory subpopulations of adipose tissue macrophages (ATMs) identified hitherto include the conventional “M1-like” CD11C-expressing ATM and the newly discovered metabolically activated CD9-expressing ATM; however, the relationship among ATM subpopulations is unclear. The ER stress sensor inositol-requiring enzyme 1α (IRE1α) is activated in the adipocytes and immune cells under obesity. It is unknown whether targeting IRE1α is capable of reversing insulin resistance and obesity and modulating the metabolically activated ATMs. We report that pharmacological inhibition of IRE1α RNase significantly ameliorates insulin resistance and glucose intolerance in male mice with diet-induced obesity. IRE1α inhibition also increases thermogenesis and energy expenditure, and hence protects against high fat diet-induced obesity. Our study shows that the “M1-like” CD11c+ ATMs are largely overlapping with but yet non-identical to CD9+ ATMs in obese white adipose tissue. Notably, IRE1α inhibition diminishes the accumulation of obesity-induced metabolically activated ATMs and “M1-like” ATMs, resulting in the curtailment of adipose inflammation and ensuing reactivation of thermogenesis, without augmentation of the alternatively activated M2 macrophage population. Our findings suggest the potential of targeting IRE1α for the therapeutic treatment of insulin resistance and obesity.

Previously, we demonstrated that specific knockout of Sirtuin 3 (Sirt3) in endothelial cells (ECs) impairs coronary flow reserve and diastolic function in mice. So far, the role of endothelial Sirt3 in the coronary arteries remains unexplored. Male and female Sirt3 EC-knockout (Sirt3 EC KO) mice and Sirt3-Lox-P mice at 14-15-month old age were used to assess the roles of endothelial Sirt3 on the microvascular functions. Mouse coronary arteries (CA) and middle cerebral arteries (MCA) were isolated and mounted in a myograph system. Myogenic response was evaluated by increasing perfusion pressure. Ach (Acetylcholine, 1nM-100μM)-induced EC-dependent vasodilation and NO donor SNP (sodium nitroprusside,1nM-10μM)-induced EC-independent vasodilation were examined. Myogenic response was significantly impaired in both CA and MCA of male and female Sirt3 ECKO mice, as evidenced by a lack of constriction while raising perfusion pressure from 10 mmHg to 150 mmHg. Both wall thickness and cross-sectional area were increased in CA and MCA of male and female Sirt3 EC KO mice. The wall to lumen ratio was increased in MCA, but did not change in CA of Sirt3 EC KO mice, compared to SIRT3-Lox-p mice. The wall tension and myogenic tone were significantly decreased in CA and MCA of both male and female Sirt3 EC KO mice. The vessel stiffness was not significantly different between SIRT3EC KO mice and SIRT3-Lox-p mice. In CA, the Ach-induced EC-dependent vasodilation was reduced in both male and female Sirt3 ECKO mice. Intriguingly, the SNP-induced EC-independent vasodilation was significantly reduced in female Sirt3 ECKO mice, but did not observed in male Sirt3 ECKO mice. In MCA, the Ach-induced EC-dependent vasodilation was only impaired in male Sirt3 ECKO mice, but was not observed in female Sirt3 ECKO mice. Whereas, the SNP-induced EC-independent vasodilation was impaired in both male and female Sirt3 EC KO mice as compared to their control SIRT3-Lox-p mice. These data revealed a sex and organ specific role of Sirt3 in EC, i.e. there is more EC-independent dysfunction in female CA, but greater impairment of EC-dependent relaxation in male MCA. Our study demonstrates the critical role of endothelial Sirt3 in maintaining microvascular function in coronary and cerebral arteries.

P36 Loss of POU4F2/BRN3B in the stressed heart compromises the adaptive hypertrophic response in male BRN3B ko mice, but not BRN3B ko female mice

TAp63 is a transcription factor that has been reported to be important for energy and glucose homeostasis. However, where and how TAp63 functions is unclear. Chow-fed mutant mice with TAp63 deleted from pro-opiomelanocortin (POMC) neurons showed normal body weight and glucose metabolism in both sex. However, HFD fed male and female mice that lack TAp63 from POMC neurons both showed glucose intolerance (GTT) and insulin resistance independent of body weight. Strikingly, deletion of TAp63 in POMC neurons confers “male-like” diet-induced obesity (DIO) to female mice, but does not affect DIO phenotypes in male mice. In contrast, overexpression of TAp63 in POMC neurons partially prevents DIO in male mice but not in female mice. Here we show that POMC neurons in female mice display higher neural activities and express higher POMC mRNAs compared to male counterparts, and both these enhancements in female POMC neurons are correlated to higher expression levels of TAp63. Further, we found that the deletion of TAp63 reduced POMC neuronal activity and diminished the sexual dimorphism of POMC neural activity by decreasing the responsiveness of ERα-positive POMC neurons to the ERα agonist. Finally, we found that TAp63 binds to the promoter of POMC gene and regulate POMC gene expression in POMC neurons. In addition, the expression of energy homeostasis related genes like SRC-1, ERα and TAp63 target genes Sirt1, AMPKα2, LKB1 were also decreased in mice lacking TAp63 in POMC neurons. Collectively, TAp63 in mature POMC neurons regulates glucose homeostasis and TAp63 is one key molecular driver for the sexual dimorphism in energy homeostasis. Disclosure C. Wang: None. Y. He: None. P. Xu: None. Y. Yang: None. Y. Xu: None.

Thyrotropin-releasing hormone (TRH) is expressed in the brain, and a few peripheral tissues. Apart from controlling TSH secretion from the pituitary, it has an anorexic effect. TRH is a short-lived intercellular signaling molecule, hydrolyzed by the TRH-degrading ectoenzyme (TRH-DE), a narrow specificity peptidase which only known biological substrate is TRH. TRH-DE activity is mainly detected in brain, with an uneven distribution among regions; at the base of the third ventricle, Trhde is expressed by β2 tanycytes whose end-feet form synaptoid contacts with TRH terminals in the external layer of the median eminence (ME). Tanycyte TRH-DE activity is sensitive to thyroid hormone feedback and energy balance clues and may contribute to adjust the thyroid axis to changing energy needs. To test whether this enzyme is relevant for energy balance, we used mice generated in the B6/129S background in which Trhde exon 2 was deleted and backcrossed to C57BL/6NJ background for 11 generations. TRH-DE activity was reduced in heterozygote (HT) and eliminated in homozygote (KO) compared to wild type (WT) animals. Male mice (70-75 days old) were switched from a standard diet to a high fat high fructose diet (HFFD) for 9 weeks. On HHFD, KO animals ingested less kcal and gained less body weight (BW) than WT animals. Bio-impedance data indicated a lower fat mass in KO mice, compared to WT or HT mice. Glucose tolerance was higher in KO mice than in WT or HT mice. To clarify the specific relevance of tanycyte TRH-DE, we used adeno-associated virus (AAV) vectors in male rats to either knock down (with TRH-DE*, a dominant negative isoform) or overexpress TRH-DE in ME tanycytes. Serum TSH concentration increased when TRH-DE activity decreased, and the converse occurred when TRH-DE activity increased. Thus, TRH-DE activity from β2-tanycytes controls the concentration of TSH in the circulation, probably because it regulates the turnover of TRH before entry into the hypothalamus-pituitary portal vessels. However, 2- or 3-weeks treatment with AAV-TRH-DE or AAV-TRH-DE* did not affect BW. To further test the role of peripheral TRH-DE activity, a phosphinic analogue of TRH (P-TRH) was administered to adult male HFFD mice during 28 days through osmotic pumps connected to an intraperitoneal catheter. P-TRH treatment reduced TRH-DE activity in serum, but not inside the blood brain barrier, compared with mice treated with vehicle; however, treatment with P-TRH did not change food consumption and BW. In conclusion, ablation of TRH-DE impedes diet-induced obesity in male mice, possibly through enhanced thyroid axis activity and amplification of the anorectic effect of TRH. Supported in part by grants from DGAPA-UNAM (PAPIIT IN206712, IN206416, IN212719), and CONACYT (CB154931, CB254960 and PN562).

Transient receptor potential cation channel C (TRPC) is essential for hypoxic pulmonary vasoconstriction response and alveolar gas exchange. We previously showed that whole body TRCP6 deficient (TRPC6 KO) mice are obese and resistant to the anorexic effects of leptin, a hormone that modulates ventilatory control. In the present study we investigated ventilatory responses to hypercapnia (7% CO2) in male and female 22‐week‐old TRPC6 KO and B6/129 control mice fed normal or high fat diet (HFD) since weaning (n=5 per group/sex/diet). Pulmonary ventilation (VE), tidal volume (VT) and respiratory frequency (FR) were measured using the plethysmography method. TRPC6 KO mice were heavier than control mice when fed normal diet (46.2±1.9 vs. 36.6±2.2 g in males and 34.0±1.4 vs. 26.9±1.3 g in females). However, when fed a HFD, male and female TRPC6 KO and B6/129 mice had similar body weight (43.6±0.4 vs. 47.5±1.4 g and 40.1±0.6 vs. 40.7±2.5 g, respectively). Compared to B6/129 controls male and female TRPC6 KO mice fed normal diet had similar VE (1817± 233 vs. 2139±135 and 2146± 404 vs. 2943±421 ml/min/kg) and VT (10.9±0.6 vs. 9.9±0.6 and 17.0±3.4 vs. 14.6±0.8 ml/kg), but lower FR (130±9 vs. 217±14 and 127±5 vs. 201±26 breaths/min) at room air. During hypercapnia, only male TRPC6 KO mice exhibited lower VE (3631± 493 vs. 6003±698 ml/min/kg), which was due to lower FR (235 ±16 vs. 305±19 breaths/min) and VT (15.3±1.0 vs. 19.5±1.4 ml/kg) responses to 7% CO2. Although female TRPC6 KO mice also showed lower FR to hypercapnia compared to B6/129 controls (242±6 vs. 313±22 breaths/min) they exhibited higher VT (28.4±3.0 vs. 25.8±2.0 ml/kg), thus resulting in similarly increased VE (6914±847 vs. 8119±956 ml/min/kg). Male and female B6/129 mice fed a HFD had significantly reduced VE responses to 7% CO2 (4020± 332 vs. 6003±698 and 5140± 194 vs. 8119±956 ml/min/kg, respectively) that were mainly caused by reduced VT (14.4±1.3 vs. 19.5±1.4 and 18.8±0.6 vs. 25.8±2.0 ml/kg). No further reduction in in ventilatory responses to hypercapnia were observed in male or female TPRC6 KO mice fed a HFD. These results suggest that TRPC6 plays an important role in ventilatory responses to hypercapnia, albeit more profound in males than females, and that HFD significantly reduces ventilatory responses to hypercapnia in both male and female control mice, but not in TRPC6 KO mice. The mechanisms involved in the reduced ventilatory responses are still unclear but may involve reduced sensitivity to leptin evoked by HFD or TRPC6 deficiency.Support or Funding InformationNIDDK 1RO1121411, NHLBI‐PO1HL51971, NIGMS P20GM104357 and U54GM115428

Regulatory T cells (Treg) play essential roles in maintaining immune homeostasis. Resident Treg in visceral adipose tissue (VAT-Treg) decrease in male obese mice, which leads to the development of obesity-associated chronic inflammations and insulin resistance. Although gender differences in immune responses have been reported, the effects of the difference in metabolic environment on VAT-Treg are unclear. We investigated the localization of VAT-Treg in female mice in comparison with that in male mice. On a high-fat diet (HFD), VAT-Treg decreased in male mice but increased in female mice. The increase was abolished in ovariectomized and HFD-fed mice, but was restored by estrogen supplementation. The IL33 receptor ST2, which is important for the localization and maturation of VAT-Treg in males, was reduced in CD4+CD25+ T cells isolated from gonadal fat of obese mice of both genders, suggesting that a different system exists for VAT-Treg localization in females. Extensive analysis of chemokine expression in gonadal fat and adipose CD4+CD25+T cells revealed several chemokine signals related to female-specific VAT-Treg accumulation such as CCL24, CCR6, and CXCR3. Taken together, the current study demonstrated sexual dimorphism in VAT-Treg localization in obese mice. Estrogen may attenuate obesity-associated chronic inflammation partly through altering chemokine-related VAT-Treg localization in females.

The vitamin D receptor (VDR) is crucial for virtually all of vitamin D's actions and is thought to be ubiquitously expressed. We hypothesized that disruption of one allele of the VDR gene would impact bone development and would have metabolic consequences. Body composition and bone mass (BMD) in VDR heterozygous (VDR HET) mice were compared to those obtained in male and female VDR KO and WT mice at 8weeks of age. Male mice were also evaluated at 16weeks, and bone marrow mesenchymal stem cell (MSC) differentiation was evaluated in VDR female mice. Additionally, female VDR HET and WT mice received intermittent PTH treatment or vehicle (VH) for 4weeks. BMD was determined at baseline and after treatment. MRI was done in vivo at the end of treatment; μCT and bone histomorphometry were performed after killing the animals. VDR HET male mice had normal skeletal development until 16weeks of age but showed significantly less gain in fat mass than WT mice. In contrast, female VDR HET mice showed decreased total-body BMD at age 8weeks but had a normal skeletal response to PTH. MSC differentiation was also impaired in VDR HET female mice. Thus, female VDR HET mice show early impairment in bone acquisition, while male VDR HET mice exhibit a lean phenotype. Our results indicate that the VDR HET mouse is a useful model for studying the metabolic and skeletal impact of decreased vitamin D sensitivity.

Recent studies demonstrate fibroblast growth factors act peripherally and centrally to regulate energy homeostasis. Fibroblast growth factor 2 (FGF2) is exclusively expressed by astrocytes and has hypophagic and weight reducing effects. The objective of this study is to further elucidate the role of FGF2 on feeding and energy balance using FGF2 KO animals. To this end, we exposed male and female wild-type and FGF2 KO mice a regular chow or a 60% high fat diet over the course of 6 weeks. We hypothesized FGF2KO animals would exhibit hyperphagia and weight gain, and that this would augment in the presence of a high fat diet. We also hypothesized FGF2KO animals would increase carbohydrate expenditure and decrease energy expenditure, and that there would be differences between the sexes. Our results demonstrate that both WT and FGF2KO animals gained weight similarly and had similar caloric intake when ingesting high fat diet. Carbohydrate expenditure was increased in male KO's and female KO's had increased ambulatory counts during the night phase. Future studies should investigate the effects of stressed FGF2KO animals and challenge them on HFD and subject them to more behaviour testing to calculate energy expenditure and anxiety-like behaviour while challenging them on HFD. Furthermore, they should continue to explore the circuitry involved in energy balance and regulation and elucidate FGF2's role in regulating this circuitry and the HPA-axis.