Background and aimsCachexia 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.MethodsSurgical biopsies for scAT were obtained from gastrointestinal cancer patients, who were signed up into the following groups: cancer cachexia (CC, n = 11), weight‐stable cancer (WSC, n = 9) and weight‐stable control (non‐cancer) (control, n = 7). The stable weight groups were considered as those with no important weight change during the last year and body mass index <25 kg/m2. Subcutaneous AT fibrosis was quantified and characterized by quantitative PCR, histological analysis and immunohistochemistry.ResultsThe degree of fibrosis and the distribution and collagen types (I and III) were different in WSC and CC patients. CC patients showed more pronounced fibrosis in comparison with WSC. Infiltrating macrophages surrounding adipocytes and CD3 Ly were found in the fibrotic areas of scAT. Subcutaneous AT fibrotic areas demonstrated increased monocyte chemotactic protein 1 (MCP‐1) and Cluster of Differentiation (CD)68 gene expression in cancer patients.ConclusionsOur data indicate architectural modification consisting of fibrosis and inflammatory cell infiltration in scAT as induced by cachexia in gastrointestinal cancer patients. The latter was characterized by the presence of macrophages and lymphocytes, more evident in the fibrotic areas. In addition, increased MCP‐1 and CD68 gene expression in scAT from cancer patients may indicate an important role of these markers in the early phases of cancer.

Adipose tissue is comprised of adipocytes and many other cell types which engage in dynamic crosstalk in a highly innervated and vascularized tissue matrix. Although studied for decades, it is only recently appreciated that extensive arbors of sensory and sympathetic nerve fibers play a dominant role in regulation of adipose functions. Here we summarize recent literature suggesting adipocytes signal to local sensory nerve fibers in response to perturbations in lipolysis and lipogenesis. Such adipocyte signaling to the central nervous system (CNS) causes sympathetic output to distant adipose depots and potentially other metabolic tissues to regulate systemic glucose homeostasis. Recently identified paracrine factors that mediate such adipocyte-neuron crosstalk are reviewed. Similarly, immune cells and endothelial cells within adipose tissue communicate with local nerve fibers to modulate neurotransmitter tone, blood flow, adipocyte differentiation and energy expenditure, including adipose "browning" to produce heat. This understudied field of "neurometabolism" related to adipose tissue biology has great potential to reveal new mechanistic insights and potential therapeutic strategies for obesity and type 2 diabetes.How fat is stored in mammals sparked vigorous scientific debate in the mid to late 1800s, yielding conflicting concepts and false starts. Major schools of thought proposed that fat accumulates in unspecialized connective tissue cells in the form of multilocular "mulberry" cells, or that "wandering cells" in the plasma accumulate fat and gather in the connective tissue. Another view posited the current concept that adipocytes occupy a specialized "glandular" tissue, but as late as 1901, this hypothesis was the least appealing in a comprehensive review of the literature 1 . How surprising it would be to these early investigators that adipose tissue is indeed "glandular" in the true sense, functioning as an endocrine organ that can control systemic functions 2-5 , and that it secretes potent paracrine and autocrine factors that modify its "glandular" nature 4,6,7 . It would be even more startling for these investigators to learn that adipose tissue can be a major heat generating tissue, critical for many animals to sustain body temperature during extreme cold exposure 8,9 .

BackgroundThe 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 & resultsHere 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.ConclusionsThese 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.

SUMMARY Adipocytes deficient in fatty acid synthase (iAdFASNKO) emit signals that mimic cold exposure to enhance the appearance of thermogenic beige adipocytes in mouse inguinal white adipose tissues (iWATs). Both cold exposure and iAdFASNKO upregulate the sympathetic nerve fiber (SNF) modulator Neuregulin 4 (Nrg4), activate SNFs, and require adipocyte cyclic AMP/protein kinase A (cAMP/PKA) signaling for beige adipocyte appearance, as it is blocked by adipocyte Gsα deficiency. Surprisingly, however, in contrast to cold-exposed mice, neither iWAT denervation nor Nrg4 loss attenuated adipocyte browning in iAdFASNKO mice. Single-cell transcriptomic analysis of iWAT stromal cells revealed increased macrophages displaying gene expression signatures of the alternately activated type in iAdFASNKO mice, and their depletion abrogated iWAT beiging. Altogether, these findings reveal that divergent cellular pathways are sufficient to cause adipocyte browning. Importantly, adipocyte signaling to enhance alternatively activated macrophages in iAdFASNKO mice is associated with enhanced adipose thermogenesis independent of the sympathetic neuron involvement this process requires in the cold.

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 × 10 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 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.

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 both genetic ablation and pharmacological inhibition of TLR4 were able to attenuate the main clinical markers of cachexia in mice bearing Lewis lung carcinoma (LLC). AT remodelling was not found in LLC tumor-bearing (TB) TLR4−/− mice due to reduced macrophage infiltration and adipocyte atrophy. TLR4−/− mice were also resistant to cold-induced browning of subcutaneous AT (scAT). Importantly, pharmacological inhibition of TLR4 (Atorvastatin) reproduced the main protective effect against AT remodeling found in TLR4−/− TB mice. Moreover, the treatment was effective in prolonging survival and attenuating tumor mass growth when compared to non-treated-TB animals. Furthermore, 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 target for novel anti-cachexia therapies.

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.

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a "browning" process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed "CRISPR-delivery particles," denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9-mediated gene deletion of ectopically expressed 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 -targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and, providing a potential therapeutic strategy for metabolic disease.