A new locus on chromosome 22q13.31 linked to recessive genetic epilepsy with febrile seizures plus (GEFS+) in a Tunisian consanguineous family.

BACKGROUND: Genetic epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome with extremely variable expressivity. The aim of our study was to identify the responsible locus for GEFS+ syndrome in a consanguineous Tunisian family showing three affected members, by carrying out a genome-wide single nucleotide polymorphisms (SNPs) genotyping followed by a whole-exome sequencing. We hypothesized an autosomal recessive (AR) mode of inheritance. RESULTS: Parametric linkage analysis and haplotype reconstruction identified a new unique identical by descent (IBD) interval of 527 kb, flanking by two microsatellite markers, 18GTchr22 and 15ACchr22b, on human chromosome 22q13.31 with a maximum multipoint LOD score of 2.51. Our analysis was refined by the use of a set of microsatellite markers. We showed that one of them was homozygous for the same allele in all affected individuals and heterozygous in healthy members of this family. This microsatellite marker, we called 17ACchr22, is located in an intronic region of TBC1D22A gene, which encodes a GTPase activator activity. Whole-exome sequencing did not reveal any mutation on chromosome 22q13.31 at the genome wide level. CONCLUSIONS: Our findings suggest that TBC1D22A is a new locus for GEFS+.

Relationship of thyroid function with obesity and type 2 diabetes in euthyroid Tunisian subjects.

OBJECTIVES: Although a relationship between obesity and metabolic consequences with thyroid function has been reported, the underlying pathogenesis is not completely known. In the current study, we evaluated the thyroid function in obese and/or diabetic patients compared to healthy normal weight peers, exploring the possible association between components of metabolic syndrome and thyroid function parameters. METHODS: We recruited 108 subjects (56 male and 52 female). In all subjects, thyroid stimulating hormone (TSH), free thyroxine (FT4), fasting plasma levels of insulin and glucose, homeostasis model assessment for insulin resistance, and obesity parameters were assessed. RESULTS: We found that circulating levels of TSH and FT4 were significantly increased in overweight and obese subjects. However, the data do not reveal any change of these hormones in diabetics. Multivariate linear regression analysis showed that TSH was directly associated with both obesity and insulin resistance parameters (p < 0.05). FT4 was negatively associated only with obesity parameters (p < 0.05). CONCLUSIONS: Our data strongly support that the changes of thyroid hormones may be influenced by adiposity and its metabolic consequences, such as insulin resistance. This relationship can be explained by a cross talk between adipose tissue release and thyroid function. Nevertheless, metformin treatment seems to affect thyroid function in diabetic patients by maintaining plasma thyrotropin levels to subnormal levels.

The effect of tobacco, XPC, ERCC2 and ERCC5 genetic variants in bladder cancer development.

BACKGROUND: In this work, we have conducted a case-control study in order to assess the effect of tobacco and three genetic polymorphisms in XPC, ERCC2 and ERCC5 genes (rs2228001, rs13181 and rs17655) in bladder cancer development in Tunisia. We have also tried to evaluate whether these variants affect the bladder tumor stage and grade. METHODS: The patients group was constituted of 193 newly diagnosed cases of bladder tumors. The controls group was constituted of non-related healthy subjects. The rs2228001, rs13181 and rs17655 polymorphisms were genotyped using a polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: Our data have reported that non smoker and light smoker patients (1-19PY) are protected against bladder cancer development. Moreover, light smokers have less risk for developing advanced tumors stage. When we investigated the effect of genetic polymorphisms in bladder cancer development we have found that ERCC2 and ERCC5 variants were not implicated in the bladder cancer occurrence. However, the mutated homozygous genotype for XPC gene was associated with 2.09-fold increased risk of developing bladder cancer compared to the control carrying the wild genotype (p = 0.03, OR = 2.09, CI 95% 1.09-3.99). Finally, we have found that the XPC, ERCC2 and ERCC5 variants don't affect the tumors stage and grade. CONCLUSION: These results suggest that the mutated homozygous genotype for XPC gene was associated with increased risk of developing bladder. However we have found no association between rs2228001, rs13181 and rs17655 polymorphisms and tumors stage and grade.

In vivo prevention of bladder urotoxicity: purified hydroxytyrosol ameliorates urotoxic effects of cyclophosphamide and buthionine sulfoximine in mice.

Urotoxicity is a troublesome complication associated with cyclophosphamide (CP) and L-buthionine-SR-sulfoximine (BSO) treatment in chemotherapy. With this concern in mind, the present study investigated the potential effects of a hydroxytyrosol extract from olive mill waste (OMW) on urotoxicity induced by acute CP and BSO doses using a Swiss albino mouse model. Toxicity modulation was evaluated by measuring lipid peroxidation (LPO) and antioxidants in urinary bladder. The findings revealed that the hydroxytyrosol extract exerted a protective effect not only on LPO but also on enzymatic antioxidants. When compared to the controls, the CP-treated animals underwent significant decreases in the glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GP), and catalase (CAT) activities. The level of glutathione (GSH) was also reduced with increased doses of LPO in the CP-treated animals. L-Buthionine-SR-sulfoximine treatment exerted an additive toxic effect on the CP-treated animals. Interestingly, pretreatment with the hydroxytyrosol extract restored the activities of all enzymes back to normal levels and exhibited an overall protective effect on the CP- and BSO-induced toxicities in urinary bladder. The restoration of GSH through the treatment with the hydroxytyrosol extract can play an important role in reversing CP-induced apoptosis and free radical-mediated LPO. 

Expression of WISP3 and RhoC genes at mRNA and protein levels in inflammatory and noninflammatory breast cancer in Tunisian patients.

Previous studies have shown the expression WISP3 and RhoC in cell lines of inflammatory breast cancer (IBC). The aim in the current study was to compare the expression of both genes, in biopsy samples collected from Tunisian patients with localized or metastatic breast cancer and patients with IBC. We investigated 127 patients enrolled in Salah Azaiez Institute in Tunis. Using the RT-PCR, we showed the phenotype (WISP3-, RhoC+) is significantly associated with IBC tumors, while the (WISP3+, RhoC-)phenotype is mostly associated to non-IBC tumors. The frequencies of these tumor phenotypes are significantly different between these tumor groups (p = 10(- 7); relative risk or RR = 3.25; confidential interval or CI 95% = 1.90-5.53). Immunohistochemical test revealing the presence of WISP3 and RhoC proteins correlates with the expression in the biopsy of their encoding genes as detected by RT-PCR. In conclusion, it appears that WISP3 and RhoC genes expression status defines a molecular signature of IBC.

Discrimination by RT-Nested PCR of Alpha-Lactalbumin Transcript Expression in Mammary Tumors.

Alpha-lactalbumin is a protein of milk expressed by mammary epithelial cells and by some breast tumors. The alpha-lactalbumin gene has two transcripts. The expression of transcript 2 has not yet been studied. The main objective of this paper was to establish the expression profile of alpha-lactalbumin at the mRNA level and to develop a technique discriminating between the two transcripts. The study was performed on 46 fresh mammary biopsies: 17 malignant tumors, 13 benign tumors, and 16 adjacent healthy tissues. We developed an RT-nested PCR to detect LALBA gene expression without distinction between transcripts. We also designed an RT-nested PCR that detects transcript 2. Both nested PCRs avoid amplification of potentially contaminating genomic DNA. We show that benign tumors tend to appear at a young age contrarily to malignant tumors appearing later with p value = 0.002. Moreover, LALBA transcript expression varies among tissues more important in benign (69%) than in malignant (35%) tumors with significant p value = 0.041. The intensity of the bands reflected differential expression between patients with concordant data between both RT-nested PCRs. We also performed a qualitative analysis according to histopathological parameters and molecular subtypes. The expression of transcript 2 appeared associated with the HER2+ subtype, while transcript 1 was associated with the luminal A and triple-negative subtypes. In conclusion, we designed specific and sensitive methods to detect LALBA transcripts. We show for the first time a differential expression of these transcripts, but we need to confirm their potential use as markers in breast cancer.

Induction of epithelial-mesenchymal transition (EMT) and Gli1 expression in head and neck squamous cell carcinoma (HNSCC) spheroid cultures.

Tumor microenvironment provides a specialized niche in which a population of stem-like cells is enriched and contributes to cancer progression. Moreover, cancer stem cell (CSC) phenotype has been associated with epithelial-mesenchymal transition (EMT). Here we investigated the effect of tumor microenvironment on the phenotypic characteristics of head and neck cancer cells and expression of CSC markers using a three-dimensional (3D), spheroid, culture system of CAL33 cell line from human tongue squamous cell carcinoma. CAL33 cells derived from 2D monolayer cultures were grown in spheroid cultures containing serum-free medium (epidermal growth factor [EGF], fibroblast growth factor [FGF], and insulin). Adherent CAL33 cells from spheroids or standard control cultures were grown in the presence/absence of serum in combination with hypoxia/normoxia. Markers of EMT, CSC, and hypoxia were analyzed either by Western blotting, immunofluorescence, or reverse transcription quantitative PCR. Spheroid cultures showed hypoxic microenvironment (high carbonic anhydrase IX [CAIX] expression), mesenchymal-like characteristics (reduced E-cadherin and increased vimentin and N-cadherin expression, presence of larger colonies comprised of larger, spread cells with lower density), and increased expression of the CSC marker glioma-associated oncogene homolog 1 (Gli1). These effects were recapitulated in serum-free adherent CAL33 cells maintained for prolonged periods in hypoxia (1% O2) but, in contrast, were completely abolished by the presence of serum. Overall, we found that a combination of hypoxia, EGF and FGF was essential to induce the EMT in adherent CAL33 cell cultures. The addition of serum rapidly reverts the EMT of cells, affects CSC phenotype and, thus, prevents the detection of such cells in tumor cell lines.

Impact of Metabolism on T-Cell Differentiation and Function and Cross Talk with Tumor Microenvironment.

The immune system and metabolism are highly integrated and multilevel interactions between metabolic system and T lymphocyte signaling and fate exist. Accumulating evidence indicates that the regulation of nutrient uptake and utilization in T cells is critically important for the control of their differentiation and manipulating metabolic pathways in these cells can shape their function and survival. This review will discuss some potential cell metabolism pathways involved in shaping T lymphocyte function and differentiation. It will also describe show subsets of T cells have specific metabolic requirements and signaling pathways that contribute to their respective function. Examples showing the apparent similarity between cancer cell metabolism and T cells during activation are illustrated and finally some mechanisms being used by tumor microenvironment to orchestrate T-cell metabolic dysregulation and the subsequent emergence of immune suppression are discussed. We believe that targeting T-cell metabolism may provide an additional opportunity to manipulate T-cell function in the development of novel therapeutics.

Modulation of the Type I Interferon Response Defines the Sensitivity of Human Melanoma Cells to Oncolytic Measles Virus.

BACKGROUND: Oncolytic viruses such as live-attenuated, vaccine strains of measles virus (MV) have recently emerged as promising cancer treatments, having shown significant antitumor activity against a large variety of human tumors. OBJECTIVE: Our study aims at determining which parameters define the sensitivity of human melanoma cells to oncolytic MV infection. METHODS: We analyzed both in vitro and in vivo the oncolytic activity of MV against a panel of human melanoma cell established in our laboratory. We tested whether either type I interferons or the interferon pathway inhibitor Ruxolitinib could modulate the sensitivity of these cells to oncolytic MV infection. RESULTS: Human melanoma cells exhibit varying levels of sensitivity to MV infection in culture and as tumor xenografts. As these differences are not explained by their expression level of the CD46 receptor, we hypothesized that antiviral immune responses may be suppressed in certain cell resulting in their inability to control infection efficiently. By analyzing the type I IFN response, we found that resistant cells had a fully functional pathway that was activated upon MV infection. On the contrary, sensitive cell showed defects in this pathway. When pre-treated with IFN-α and IFN-ß, all but one of the sensitive cell became resistant to MV. Cells resistant to MV were rendered sensitive to MV with Ruxolitinib. CONCLUSION: Type I interferon response is the main determinant for the sensitivity or resistance of melanoma to oncolytic MV infection. This will have to be taken into account for future clinical trials on oncolytic MV.

Intrinsic and Tumor Microenvironment-Induced Metabolism Adaptations of T Cells and Impact on Their Differentiation and Function.

It is well recognized that the immune system and metabolism are highly integrated. In this context, multilevel interactions between metabolic system and T lymphocyte signaling and fate exist. This review will discuss different potential cell metabolism pathways involved in shaping T lymphocyte function and differentiation. We will also provide a general framework for understanding how tumor microenvironmental metabolism, associated with hypoxic stress, interferes with T-cell priming and expansion. How T-cell metabolism drives T-cell-mediated immunity and how the manipulation of metabolic programing for therapeutic purposes will be also discussed.

Incidence Trend for Non-Hodgkin Lymphoma in the North Tunisian Population, 1998-2009.

BACKGROUND: In 2008, non-Hodgkin lymphoma ranked tenth among other malignancies worldwide with an incidence of around 5 cases per 100,000 in both genders. The latest available rates in Tunisia are from 2006. MATERIALS AND METHODS: This study aimed to provide an update about NHL incidence for 2009 and its trend between 1998 and 2009 as well as a projection until 2024, using data from the Salah Azaiz Institute hospital registry and the Noth Tunisia cancer registry. RESULTS: In 2009, the NHL incidence in the north of Tunisia was 4.03 cases per 100,000, 4.97 for men and 3.10 for women. Diffuse large B-cell lymphoma (DLBCL) accounted for 63.2% of all NHL subtypes. Between 1998 and 2009, the overall trend showed no significant change. When we compared the trend between two periods (1998-2005 and 2005-2009), joinpoint regression showed a significant decrease of NHL incidence in the first period with an annual percentage change (APC) of -6.7% (95% CI:[-11.2%;-2%]), then the incidence significantly increased from 2005 to 2009 with an APC of 30.5% (95% CI: [16.1%; 46.6%]. The analyses of the different subtype trends showed a significant decrease in DLBCL incidence between 1998 and 2000 (APC:-21.5; 95% CI: [-31.4%;-10.2%]) then the incidence significantly increased between 2004 and 2007 (APC: 18.5; 95% CI: [3,6%;35.5%]). Joint point analysis of the age-period-cohort model projection showed a significant increase between 2002 and 2024 with an APC of 4.5% (%95 CI: [1.5%; 7.5%]). The estimated ASR for 2024 was 4.55/100 000 (95% CI: [3.37; 6.15]). CONCLUSIONS: This study revealed an overall steady trend in the incidence of NHL in northern Tunisia between 1998 and 2009. Projection showed an increase in the incidence in NHL in both genders which draw the attention to the national and worldwide burden of this malignancy.

Y-chromosomal STR haplotypes in three ethnic groups and one cosmopolitan population from Tunisia.

The 11 Y-chromosomal short tandem repeats (STRs) included in the Promega Corporation PowerPlex Y System (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438 and DYS439) were typed in three ethnic groups ("Andalusians", Berber and Arab) and one cosmopolitan population (Tunis) from Tunisia, summing up 247 individuals, and 139 different haplotypes. Focusing the analysis on the seven Y-STRs of the YHRD Minimal Haplotype Core (DYS385 excepted), "Andalusians" showed no differences from the Cosmopolitan and the Arab samples previously published (our Arab sample presented an extremely low haplotype diversity), but were different from the Berbers. The Berbers from Tunisia were not different from those from Morocco.

Consanguinity Protecting Effect Against Breast Cancer among Tunisian Women: Analysis of BRCA1 Haplotypes.

The purpose of this study is to assess the effect of consanguinity on breast cancer incidence in Tunisia. We conducted a case-control study to evaluate the involvement of heterozygote and homozygote haplotypes of BRCA1 gene SNPs according to consanguinity among 40 cases of familial breast cancer, 46 cases with sporadic breast cancer and 34 healthy controls. We showed significant difference in consanguinity rate between breast cancer patients versus healthy controls P = 0.001. Distribution of homozygous BRCA1 haplotypes among healthy women versus breast cancer patients was significantly different; p=0.02. Parental consanguinity seems to protect against breast cancer in the Tunisian population.

MicroRNAs expression in triple negative vs non triple negative breast cancer in Tunisia: interaction with clinical outcome.

INTRODUCTION: MicroRNAs are small, non coding regulatory molecules containing approximately 21 to 25 nucleotides. They function as controllers of expression at post transcriptional levels of most human protein-coding genes and play an essential role in cell signaling pathways. The objective of the present study is to evaluate the expression profile of the following micro-RNAs: miR-10b, miR-17, miR-21, miR-34a, miR-146a, miR-148a and miR-182, and to determine their possible interaction in triple-negative and non triple-negative primary breast cancers based on clinical outcome. METHODS: 60 triple-negative and non triple-negative breast cancer cases, along with their corresponding normal samples were investigated in relation to the expression of the seven studied miRNAs using qPCR Syber Green. RESULTS: We observed that miR-21, miR-146a and miR-182 were significantly over expressed in triple negative breast cancer. Moreover, miR-10b, miR-21 and miR-182 were significantly associated to lymph node metastases occurrence in triple negative breast carcinoma while only miR-10b was associated with grade III in non triple negative breast cancer cases. Almost all the analyzed microRNAs were strongly associated with patients' genico-obstetric history in non triple negative breast cancer cases except for miR-34a. All the studied microRNAs were strongly correlated with the use of the contraceptive pills in non triple negative breast cancer groups. The additive effect of hormonal factors in triple negative breast cancer cases showed an association with all the studied miRs except for miR-34 and miR-146a. CONCLUSION: The studied microRNAs are strongly influenced by environmental factors especially with hormonal patients' history. Moreover, miR-10b, miR-21 and miR-182 could be defined as biomarkers in breast cancer to predict both lymph node metastases and grade III occurrence.

Wild-type genotypes of BRCA1 gene SNPs combined with micro-RNA over-expression in mammary tissue leading to familial breast cancer with an increased risk of distant metastases' occurrence.

Germ line deleterious mutations of BRCA1 gene are not the unique factor that could inactivate BRCA1 protein which leads to familial breast cancer onset with distant metastases' occurrence. The present research explores the role that could be assigned to BRCA1 SNPs to inactivate BRCA1 protein and therefore to the occurrence of familial breast cancer with an increased risk of distant metastases' occurrence. The presence or the absence of BRCA1 protein was first analyzed by applying the immunohistochemistry technique to the tumors with sporadic and familial breast cancer. Then, a case-control study was conducted including 40 patients with familial breast cancer, 46 ones with sporadic breast cancer and 34 healthy controls based on the genotyping of nine BRCA1 SNPs (c.442.58delT, c.2082C>T, c.2311T>C, c.2612C>T, c.3113A>G, c.3119G>A, c.3548A>G, c.4308T>C and 4837A>G) via direct sequencing. Finally, the functional role that could be assigned to these SNPs was focused upon. miRbase site was used as a bioinformatics tool to predict potential micro-RNAs (miRs) targeting SNPs that are associated with familial breast cancer according to the results of this research. These predicted miRs were confirmed by Q-PCR analysis and correlated with BRCA1 protein expression among patients along with potential distant metastases. Clinical outcome showed that distant metastasis concerned 45 % of familial breast cancer patients and 19.5 % with sporadic breast cancer. Analysis of BRCA1 protein expression revealed a negative staining among 46.6 % of familial breast cancer patients and only 16.6 % within sporadic breast cancer ones. The association of four variants was identified within BRCA1 gene (c.442.58 delT, c.2311T>C, c.2612C>T and c.4308T>C) to familial breast cancer across their wild genotypes. miR-1179 was selected as potential miR that targets the region of BRCA1 mRNA containing the c.2311T>C variant within the TT genotype. The expression of miR-1179 was significantly associated with familial breast cancer patients without BRCA1 deleterious mutations compared to those with sporadic breast cancer according to TT genotype along with BRCA1 negative staining and according to the occurrence of distant metastases. Combination between TT genotype of c.2311T>C and miR-1179 over-expression could generate a lack of BRCA1 protein leading to a high risk of familial breast cancer with distant metastases.

Human subcutaneous adipose tissue Glut 4 mRNA expression in obesity and type 2 diabetes.

Cellular resistance to insulin caused by reduced glucose transport and metabolism is a primary defect leading to the development of metabolic disease. While the etiology of insulin resistance is multifactorial, reduced insulin action is associated with impaired activity of the glucose transporter GLUT4 in insulin-sensitive tissues. Yet, the role of adipose tissue GLUT4 deregulation in the pathogenesis of insulin resistance, obesity, and diabetes is still unclear. In this study, we assessed the relative GLUT4 level in human subcutaneous adipose tissue from obese, diabetic, and diabetic obese versus control subjects, using a real-time PCR method. GLUT4 mRNA levels were considerably decreased among type 2 diabetic patients compared with those of the controls (P < 0.01), whereas no such difference was found between obese and normal-weight controls. Multiple linear regressions analysis in both diabetic non-obese and diabetic obese groups showed a negative correlation between GLUT4 mRNA expression and both markers of obesity or insulin resistance (P < 0.01). However, in obese group, GLUT4 was inversely associated only with HOMA-IR (P < 0.01). Our findings showed that adipose GLUT4 gene expression changes were more related to insulin resistance and type 2 diabetes rather than to obesity.

Aurora A overexpression and pVHL reduced expression are correlated with a bad kidney cancer prognosis.

We investigate the expression and localization of the tumor suppressor protein pVHL as well as the oncoprotein Aurora A kinase in kidney cancer. Both Aurora A kinase and pVHL protein status were evaluated using immunohistochemistry. The Aurora A expression is correlated with the Fuhrman grade and the TNM stage, while the pVHL expression is correlated with the capsule rupture and the TNM stage. Aurora A kinase expression increases in malignant tissue comparing to the non-malignant one. And there is a decrease in pVHL expression from the adjacent healthy tissues to the tumor`s ones. The two kinds of opposite tumor profiles display significant distribution difference according to TNM stages. It could be proposed that the absence of Aurora A protein associated with a strong expression of pVHL in clear cells kidney carcinoma are of good prognosis for the disease.

Smoking and polymorphisms in folate metabolizing genes and their effects on the histological stage and grade for bladder tumors.

Folates are the common sources of DNA synthesis and methylation. Cigarette smoking and genetic susceptibility of folate enzymes are two suspected factors most closely associated with bladder cancer development. This study sought to determine the effect of smoking and genetic polymorphisms in folate metabolizing enzymes on the histological stage and grade of bladder tumors in Tunisian patients. A total of 130 patients with urothelial cell carcinomas were examined with respect to smoking status, MTHFR (5,10-methylenetetrahydrofolate reductase), MTR (methionine synthase), MTRR (methionine synthase reductase) and TYMS (thymidylate synthase) genotypes distribution. Our data have reported that tobacco, MTHFR, MTR and MTRR genotypes were not associated with bladder tumor stage. Only TYMS 3R*G/3R*C genotype was associated with increased risk of developing invasive tumors compared to reference group (RR = 1.74; 95% CI: 0.97-3.12). When we studied the superficial bladder tumor group, we have shown a significant statistical differences for the TYMS 3R*G/2R genotype. This genotype presented a 1.68-fold increased risk of developing high grade tumors compared to reference group (RR = 1.68; 95% CI: 1.12-2.54). Moreover, we have shown that patients having at least one copy of 2R allele were at 4.23-fold increased risk for developing high grade tumors compared to reference group (P = 0.022).

Peroxisome proliferator-activated receptor-gamma (PPARgamma) modulates hypothalamic Trh regulation in vivo.

Thyroid hormone receptor (TR) and peroxisome proliferator-activated receptor gamma (PPARgamma) co-regulate numerous peripheral metabolic responses. To examine potential crosstalk between PPARgamma and TRbeta in the hypothalamus, thyrotropin-releasing hormone (Trh) regulation in the newborn mouse hypothalamus was followed. QPCR showed PPARgamma to be expressed in the hypothalamus at this developmental stage. Intracerebral injection of PPARgamma agonists modified transcription from a TRH-luc construct introduced into the hypothalamus and increased serum thyroxine levels. Furthermore, shRNA-based in vivo PPARgamma knockdown amplified T(3)-independent transcription and PPARgamma overexpression dose-dependently abrogated T(3)-dependent Trh repression. Overexpression of retinoid X receptor-alpha (RXRalpha), the common heterodimeric partner of PPARgamma and TRbeta, rescued PPARgamma abrogation of T(3)-dependent repression. Thus, competition for RXR could represent one mechanism underlying this hypothalamic crosstalk between PPARgamma and TRbeta. These demonstrations of PPARgamma effects on hypothalamic Trh transcription in vivo consolidate the role of the TRH neuron as a central integrator of energy homeostasis.