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 anticancer 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 (2x10(7)) 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 -80oC (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-gamma, adiponectin, LPL and C/EBP-alpha 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Mogi das Cruzes, Integrated Grp Biotechnol, Lab Adipose Tissue Biol, Mogi Das Cruzes, BrazilUniv São Paulo, Inst Biomed Sci, Canc Metab Res Grp, São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Physiol Lab, São Paulo, BrazilUniv Estadual Maringa, Dept Physiol Sci, Maringa, Parana, BrazilUniversidade Federal de São Paulo, Dept Biomed Engn, Sao Jose Dos Campos, BrazilBoston Sch Med, Dept Biochem, Boston, MA USAUniversidade Federal de São Paulo, Dept Biomed Engn, Sao Jose Dos Campos, BrazilFAPESP: 2010/51078-1FAPESP: 2008/54091-9FAPESP: 2012/51094-1Web of Scienc

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

Abstract 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

Crosstalk between corepressor NRIP1 and cAMP signaling on adipocyte thermogenic programming

Objectives: Nuclear receptor interacting protein 1 (NRIP1) suppresses energy expenditure via repression of nuclear receptors, and its depletion markedly elevates uncoupled respiration in mouse and human adipocytes. We tested whether NRIP1 deficient adipocytes implanted into obese mice would enhance whole body metabolism. Since β-adrenergic signaling through cAMP strongly promotes adipocyte thermogenesis, we tested whether the effects of NRIP1 knock-out (NRIP1KO) require the cAMP pathway. Methods: NRIP1KO adipocytes were implanted in recipient high-fat diet (HFD) fed mice and metabolic cage studies conducted. The Nrip1 gene was disrupted by CRISPR in primary preadipocytes isolated from control vs adipose selective GsαKO (cAdGsαKO) mice prior to differentiation to adipocytes. Protein kinase A inhibitor was also used. Results: Implanting NRIP1KO adipocytes into HFD fed mice enhanced whole-body glucose tolerance by increasing insulin sensitivity, reducing adiposity, and enhancing energy expenditure in the recipients. NRIP1 depletion in both control and GsαKO adipocytes was equally effective in upregulating uncoupling protein 1 (UCP1) and adipocyte beiging, while β-adrenergic signaling by CL 316,243 was abolished in GsαKO adipocytes. Combining NRIP1KO with CL 316,243 treatment synergistically increased Ucp1 gene expression and increased the adipocyte subpopulation responsive to beiging. Estrogen-related receptor α (ERRα) was dispensable for UCP1 upregulation by NRIPKO. Conclusions: The thermogenic effect of NRIP1 depletion in adipocytes causes systemic enhancement of energy expenditure when such adipocytes are implanted into obese mice. Furthermore, NRIP1KO acts independently but cooperatively with the cAMP pathway in mediating its effect on adipocyte beiging

Body weight and food intake for different time points along the progression of cachexia.

<p>Rats were inoculated with Walker 256 tumor cells (2×10⁷ cells) or vehicle and treated daily with 5 mg of PGZ (TP5) or PBS (TC) during 26 days. (A) Body weight was measured on days 4, 7, 14 and 26 and (B) Relative body weight loss on days 7, 14 and 26-post tumor cells injection. (C) Food intake was measured daily throughout the study and (D) Tumor mass was measured on days 7, 14 and 26-post tumor cells inoculation. * p<0.05, one-way ANOVA followed by Bonferroni’s post-hoc (time from day 0), and ^# P<0.05 vs. TC.</p

Visceral adipose tissue mass during the progression of cachexia.

<p>Rats were inoculated with Walker 256 tumor cells (2×10⁷ cells) or vehicle and treated daily with 5 mg of PGZ (TP5) or PBS (TC) during 14 days. (A) RPAT and (C) MEAT tissues from tumor-bearing rats were removed and weighed on day 7 and day 14. (B) Spearman correlation between relative RPAT mass on day 7 (early-stage) and tumor growth on day 14 (cachexia-stage), from TP5. (D) Real-time PCR analysis of RNA isolated from RPAT (isolated adipocytes) in the 7th on day after tumor cell inoculation. mRNA levels of target genes were normalized to 18S. Values are mean ± s.e.m. for five to seven animals per group. RPAT-retroperitoneal adipose tissue; MEAT-Mesenteric adipose tissue. ^#p<0.05 vs. TC.</p

2-Deoxy-D[2,6 ³H]glucose uptake assay.

<p>Rats were inoculated with Walker 256 tumor cells (2×10⁷ cells) or vehicle and treated daily with 5 mg of PGZ (TP5) or PBS (TC) during 7 days. Isolated adipocytes from RPAT tissues were stimulated with 0 (basal), 2.5 and 10 (maximal) nmol/cm² of cell surface area. RPAT-retroperitoneal adipose tissue; * p<0.05, one-way ANOVA followed by Bonferroni’s post-hoc (time from day 0), ^# P<0.05 vs. TC, and ^& P<0.05 vs. Baseline Control.</p

Biochemical parameters from tumor-bearing animals during PGZ treatment.

<p>Values are mean <b>±</b>s.e.m. for five to seven animals per group.</p><p>* p<0.05, one-way ANOVA followed by Bonferroni’s post-hoc (day 7 vs. 14)</p><p>^# P<0.05 vs. TC,</p><p>^& P<0.01 vs. Baseline Control.</p><p>Biochemical parameters from tumor-bearing animals during PGZ treatment.</p

Survival of tumor-bearing animals during PGZ treatment period.

<p>To evaluate dose-response drug effect, animals were treated with four doses of PGZ normalized by rat’s body weight, which were administered daily: 5, 10, 20 and 40 mg/kg/day. Plots of Kaplan-Mayer product limit estimates of survival of a group of tumor-bearing animals receiving PGZ therapy during 26 days, ^# p<0.05 vs. TC.</p