Felipe Henriques

Cachexia-associated adipose tissue morphological rearrangement in gastrointestinal cancer patients.

Cachexia is a syndrome characterized by marked involuntary loss of body weight. Recently, adipose tissue (AT) wasting has been shown to occur before the appearance of other classical cachexia markers. We investigated the composition and rearrangement of the extracellular matrix, adipocyte morphology and inflammation in the subcutaneous AT (scAT) pad of gastrointestinal cancer patients.

Pioglitazone Treatment Increases Survival and Prevents Body Weight Loss in Tumor–Bearing Animals: Possible Anti-Cachectic Effect

Cachexia is a multifactorial syndrome characterized by profound involuntary weight loss, fat depletion, skeletal muscle wasting, and asthenia; all symptoms are not entirely attributable to inadequate nutritional intake. Adipose tissue and skeletal muscle loss during cancer cachexia development has been described systematically. The former was proposed to precede and be more rapid than the latter, which presents a means for the early detection of cachexia in cancer patients. Recently, pioglitazone (PGZ) was proposed to exhibit anti-cancer properties, including a reduction in insulin resistance and adipose tissue loss; nevertheless, few studies have evaluated its effect on survival. For greater insight into a potential anti-cachectic effect due to PGZ, 8-week-old male Wistar rats were subcutaneously inoculated with 1 mL (2×107) of Walker 256 tumor cells. The animals were randomly assigned to two experimental groups: TC (tumor + saline-control) and TP5 (tumor + PGZ/5 mg). Body weight, food ingestion and tumor growth were measured at baseline and after removal of tumor on days 7, 14 and 26. Samples from different visceral adipose tissue (AT) depots were collected on days 7 and 14 and stored at -80o C (5 to 7 animals per day/group). The PGZ treatment showed an increase in the survival average of 27.3% (P< 0.01) when compared to TC. It was also associated with enhanced body mass preservation (40.7 and 56.3%, p< 0.01) on day 14 and 26 compared with the TC group. The treatment also reduced the final tumor mass (53.4%, p<0.05) and anorexia compared with the TC group during late-stage cachexia. The retroperitoneal AT (RPAT) mass was preserved on day 7 compared with the TC group during the same experimental period. Such effect also demonstrates inverse relationship with tumor growth, on day 14. Gene expression of PPAR-γ, adiponectin, LPL and C/EBP-α from cachectic rats was upregulated after PGZ. Glucose uptake from adipocyte cells (RPAT) was entirely re-established due to PGZ treatment. Taken together, the results demonstrate beneficial effects of PGZ treatment at both the early and final stages of cachexia.

Adipocyte lipid synthesis coupled to neuronal control of thermogenic programming

Background The de novo biosynthesis of fatty acids (DNL) through fatty acid synthase (FASN) in adipocytes is exquisitely regulated by nutrients, hormones, fasting, and obesity in mice and humans. However, the functions of DNL in adipocyte biology and in the regulation of systemic glucose homeostasis are not fully understood. Methods & results Here we show adipocyte DNL controls crosstalk to localized sympathetic neurons that mediate expansion of beige/brite adipocytes within inguinal white adipose tissue (iWAT). Induced deletion of FASN in white and brown adipocytes of mature mice (iAdFASNKO mice) enhanced glucose tolerance, UCP1 expression, and cAMP signaling in iWAT. Consistent with induction of adipose sympathetic nerve activity, iAdFASNKO mice displayed markedly increased neuronal tyrosine hydroxylase (TH) and neuropeptide Y (NPY) content in iWAT. In contrast, brown adipose tissue (BAT) of iAdFASNKO mice showed no increase in TH or NPY, nor did FASN deletion selectively in brown adipocytes (UCP1-FASNKO mice) cause these effects in iWAT. Conclusions These results demonstrate that downregulation of fatty acid synthesis via FASN depletion in white adipocytes of mature mice can stimulate neuronal signaling to control thermogenic programming in iWAT.

Cold-sensing TRPM8 channel participates in circadian control of the brown adipose tissue

Transient receptor potential (TRP) channels are known to regulate energy metabolism, and TRPM8 has become an interesting player in this context. Here we demonstrate the role of the cold sensor TRPM8 in the regulation of clock gene and clock controlled genes in brown adipose tissue (BAT). We investigated TrpM8 temporal profile in the eyes, suprachiasmatic nucleus and BAT; only BAT showed temporal variation of TrpM8 transcripts. Eyes from mice lacking TRPM8 lost the temporal profile of Per1 in LD cycle. This alteration in the ocular circadian physiology may explain the delay in the onset of locomotor activity in response to light pulse, as compared to wild type animals (WT). Brown adipocytes from TrpM8 KO mice exhibited a larger multilocularity in comparison to WT or TrpV1 KO mice. In addition, Ucp1 and UCP1 expression was significantly reduced in TrpM8 KO mice in comparison to WT mice. Regarding circadian components, the expression of Per1, Per2, Bmal1, Pparα, and Pparβ oscillated in WT mice kept in LD, whereas in the absence of TRPM8 the expression of clock genes was reduced in amplitude and lack temporal oscillation. Thus, our results reveal new roles for TRPM8 channel: it participates in the regulation of clock and clock-controlled genes in the eyes and BAT, and in BAT thermogenesis. Since disruption of the clock machinery has been associated with many metabolic disorders, the pharmacological modulation of TRPM8 channel may become a promising therapeutic target to counterbalance weight gain, through increased thermogenesis, energy expenditure, and clock gene activation.

Disruption of beta3 adrenergic receptor increases susceptibility to DIO in mouse.

The brown adipose tissue (BAT) mediates adaptive changes in metabolic rate by responding to the sympathetic nervous system through β-adrenergic receptors (AR). Here, we wished to define the role played by the ARβ3 isoform in this process. This study focused on the ARβ3 knockout mice (ARβ3KO), including responsiveness to cold exposure, diet-induced obesity, intolerance to glucose, dyslipidaemia and lipolysis in white adipose tissue (WAT). ARβ3KO mice defend core temperature during cold exposure (4°C for 5 h), with faster BAT thermal response to norepinephrine (NE) infusion when compared with wild-type (WT) mice. Despite normal BAT thermogenesis, ARβ3KO mice kept on a high-fat diet (HFD; 40% fat) for 8 weeks exhibited greater susceptibility to diet-induced obesity, markedly increased epididymal adipocyte area with clear signs of inflammation. The HFD-induced glucose intolerance was similar in both groups but serum hypertriglyceridemia and hypercholesterolemia were less intense in ARβ3KO animals when compared with WT controls. Isoproterenol-induced lipolysis in isolated white adipocytes as assessed by glycerol release was significantly impaired in ARβ3KO animals despite normal expression of key proteins involved in lipid metabolism. In conclusion, ARβ3 inactivation does not affect BAT thermogenesis but increases susceptibility to diet-induced obesity by dampening WAT lipolytic response to adrenergic stimulation.

Cancer cachexia differentially regulates visceral adipose tissue turnover

Cancer cachexia (CC) is a progressive metabolic syndrome that is marked by severe body weight loss. Metabolic disarrangement of fat tissues is a very early event in CC, followed by adipose tissue (AT) atrophy and remodelling. However, there is little information regarding the possible involvement of cellular turnover in this process. Thus, in this study, we evaluated the effect of CC on AT turnover and fibrosis of mesenteric (MEAT) and retroperitoneal (RPAT) adipose tissue depots as possible factors that contribute to AT atrophy. CC was induced by a subcutaneous injection of Walker tumour cells (2 × 107) in Wistar rats, and control animals received only saline. The experimental rats were randomly divided into four experimental groups: 0 days, 4 days, 7 days and 14 days after injection. AT turnover was analysed according to the Pref1/Adiponectin ratio of gene expression from the stromal vascular fraction and pro-apoptotic CASPASE3 and CASPASE9 from MEAT and RPAT. Fibrosis was verified according to the total collagen levels and expression of extracellular matrix genes. AT turnover was verified by measurements of lipolytic protein expression. We found that the Pref1/Adiponectin ratio was decreased in RPAT (81.85%, P < 0.05) with no changes in MEAT compared with the respective controls. CASPASE3 and CASPASE9 were activated on day 14 only in RPAT. Collagen was increased on day 7 in RPAT (127%) and MEAT (4.3-fold). The Collagen1A1, Collagen3A1, Mmp2 and Mmp9 mRNA levels were upregulated only in MEAT in CC. Lipid turnover was verified in RPAT and was not modified in CC. We concluded that the results suggest that CC affects RPAT cellular turnover, which may be determinant for RPAT atrophy.

Early suppression of adipocyte lipid turnover induces immunometabolic modulation in cancer cachexia syndrome

Cancer cachexia is a multifactorial syndrome characterized by body weight loss, atrophy of adipose tissue (AT) and systemic inflammation. However, there is limited information regarding the mechanisms of immunometabolic response in AT from cancer cachexia. Male Wistar rats were inoculated with 2 × 107 of Walker 256 tumor cells [tumor bearing (TB) rats]. The mesenteric AT (MeAT) was collected on d 0, 4, 7 (early stage), and 14 (cachexia stage) after tumor cell injection. Surgical biopsies for MeAT were obtained from patients who had gastrointestinal cancer with cachexia. Lipolysis showed an early decrease in glycerol release in TB d 4 (TB4) rats in relation to the control, followed by a 6‐fold increase in TB14 rats, whereas de novo lipogenesis was markedly lower in the incorporation of glucose into fatty acids in TB14 rats during the development of cachexia. CD11b and CD68 were positive in TB7 and TB14 rats, respectively. In addition, we found cachexia stage results similar to those of animals in MeAT from patients: an increased presence of CD68+, iNOS2+, TNFα+, and HSL+ cells. In summary, translational analysis of MeAT from patients and an animal model of cancer cachexia enabled us to identify early disruption in Adl turnover and subsequent inflammatory response during the development of cancer cachexia.—Henriques, F. S., Sertié, R. A. L., Franco, F. O., Knobl, P., Neves, R. X., Andreotti, S., Lima, F. B., Guilherme, A., Seelaender, M., Batista, M. L., Jr. Early suppression of adipocyte lipid turnover induces immunometabolic modulation in cancer cachexia syndrome. FASEB J. 31, 1976–1986 (2017). www.fasebj.org

Neuronal modulation of brown adipose activity through perturbation of white adipocyte lipogenesis

Objective Crosstalk between adipocytes and local neurons may be an important regulatory mechanism to control energy homeostasis. We previously reported that perturbation of adipocyte de novo lipogenesis (DNL) by deletion of fatty acid synthase (FASN) expands sympathetic neurons within white adipose tissue (WAT) and stimulates the appearance of “beige” adipocytes. Here we tested whether WAT DNL activity can also influence neuronal regulation and thermogenesis in brown adipose tissue (BAT). Methods and results Induced deletion of FASN in all adipocytes in mature mice (iAdFASNKO) enhanced sympathetic innervation and neuronal activity as well as UCP1 expression in both WAT and BAT. This increased sympathetic innervation could be observed at both 22 °C and 30 °C, indicating it is not a response to heat loss but rather adipocyte signaling. In contrast, selective ablation of FASN in brown adipocytes of mice (iUCP1FASNKO) failed to modulate sympathetic innervation and the thermogenic program in BAT. Surprisingly, DNL in brown adipocytes was also dispensable in maintaining euthermia when UCP1FASNKO mice were cold-exposed. Conclusion These results indicate that DNL in white adipocytes influences long distance signaling to BAT, which can modify BAT sympathetic innervation and expression of genes involved in thermogenesis.

CRISPR delivery particles for developing therapeutic strategies in metabolic disease

RNA-guided engineered nucleases derived from a prokaryotic adaptive immune system known as CRISPR-Cas represent a promising platform for gene deletion and editing. As a therapeutic approach, direct delivery of Cas9 protein and guide RNA could circumvent the safety problems associated with plasmid delivery and therefore represents an attractive tool for genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fat oxidation and secretion of bioactive factors through a “browning” process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed novel CRISPR delivery particles, denoted CriPs, composed of nano-size complexes of Cas9 protein and single guide (sg)RNA, coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9 mediated gene deletion of ectopically expressed Green fluorescent protein (GFP) by CriPs was achieved in multiple cell types including a macrophage cell line, primary macrophages and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP expressing mice following intraperitoneal injection of CriPs containing sgRNA targeting Gfp. Furthermore, the disruption of the Nrip1 gene in white adipocytes by CriPs enhanced adipocyte “browning” with a marked increase of UCP1 expression. Deletion of Nrip1 by CriPs did not produce detectable off-target effects. Thus CriPs represent a novel CRISPR delivery system for Cas9 and sgRNA that is effective for ablating targeted gene products in cultured cells and in vivo, and provide a potential therapeutic strategy for metabolic disease.

Toll-Like Receptor-4 Disruption Suppresses Adipose Tissue Remodeling and Increases Survival During Cancer Cachexia Syndrome

Cancer-induced cachexia, characterized by systemic inflammation, body weight loss, adipose tissue (AT) remodeling and muscle wasting, is a malignant metabolic syndrome with undefined etiology. Here, we show that Toll-like receptor 4 (TLR4) mediates AT remodeling, in particular, AT browning and inflammatory response in mice bearing Lewis lung carcinoma (LLC). LLC tumor-bearing (TB) TLR4−/− mice were spared from AT remodeling due to a reduced macrophage infiltration and adipocyte atrophy. TLR4−/− mice were also resistant to cold-induced browning of subcutaneous AT (scAT). Importantly, pharmacological inhibition of TLR4 reproduced the main protective effect against AT remodeling found in TLR4−/− TB mice. Moreover, the treatment was effective in prolonging the survival and attenuating tumor mass growth when compared to non-treated-TB animals. Further, tumor-induced elevation of circulating pro-inflammatory cytokines was similarly abolished in both genetic ablation and pharmacological inhibition of TLR4. These data suggest that TLR4 is a critical mediator and a promising therapeutic target for cancer-induced AT remodeling. HIGHLIGHTS Genetic ablation and pharmacological inhibition of TLR4 attenuate adipose tissue remodeling during cancer-associated cachexia; TLR4 suppression play an essential role in the browning phenotype induced by cachexia; Administration of TLR4 drug inhibitor increase survival and reduces tumor mass growth in tumor bearing mice; TLR4 pathway is a promising target for cancer-cachexia therapeutic intervention.