Involvement of the gut microbiota in cancer cachexia.

Cancer cachexia, or the unintentional loss of body weight in patients with cancer, is a multiorgan and multifactorial syndrome with a complex and largely unknown etiology; however, metabolic dysfunction and inflammation remain hallmarks of cancer-associated wasting. Although cachexia manifests with muscle and adipose tissue loss, perturbations to the gastrointestinal tract may serve as the frontline for both impaired nutrient absorption and immune-activating gut dysbiosis. Investigations into the gut microbiota have exploded within the past two decades, demonstrating multiple gut-tissue axes; however, the link between adipose and skeletal muscle wasting and the gut microbiota with cancer is only beginning to be understood. Furthermore, the most used anticancer drugs (e.g. chemotherapy and immune checkpoint inhibitors) negatively impact gut homeostasis, potentially exacerbating wasting and contributing to poor patient outcomes and survival. In this review, we 1) highlight our current understanding of the microbial changes that occur with cachexia, 2) discuss how microbial changes may contribute to adipose and skeletal muscle wasting, and 3) outline study design considerations needed when examining the role of the microbiota in cancer-induced cachexia.

Decreased skeletal muscle intramyocellular lipid droplet-mitochondrial contact contributes to myosteatosis in cancer cachexia.

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice (n = 20) were implanted with LLC cells (106) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size (R2 = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle (P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia.NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.

miR155 deficiency reduces breast tumor burden in the MMTV-PyMT mouse model.

miRNA155 (miR155) has emerged as an important regulator of breast cancer (BrCa) development. Studies have consistently noted an increase in miR155 levels in serum and/or tissues in patients with BrCa. However, what is less clear is whether this increase in miR155 is a reflection of oncogenic or tumor suppressive properties. To study the effects of miR155 in a transgenic model of BrCA, we developed an MMTV-PyMT mouse deficient in miR155 (miR155-/- PyMT). miR155-/- mice (n = 11) exhibited reduced tumor number and volume palpations at ∼14-18 wk of age compared with miR155 sufficient littermates (n = 12). At 19 wk, mammary glands were excised from tumors for RT-PCR, and tumors were counted, measured, and weighed. miR155-/- PyMT mice exhibited reduced tumor volume, number, and weight, which was confirmed by histopathological analysis. There was an increase in apoptosis with miR155 deficiency and a decrease in proliferation. As expected, miR155 deficiency resulted in upregulated gene expression of suppressor of cytokine signaling 1 (Socs1)-its direct target. There was a reduction in gene expression of macrophage markers (CD68, Adgre1, Itgax, Mrc1) with miR-155-/- and this was confirmed with immunofluorescence staining for F4/80. miR155-/- increased expression of M1 macrophage marker Nos2 and reduced expression of M2 macrophage markers IL-10, IL-4, Arg1, and MMP9. Overall, miR155 deficiency reduced BrCA and improved the tumor microenvironment through the reduction of genes associated with protumorigenic processes. However, given the inconsistencies in the literature, additional studies are needed before any attempts are made to harness miR155 as a potential oncogenic or tumor suppressive miRNA.NEW & NOTEWORTHY To examine the effects of miR155 in a transgenic model of breast cancer, we developed an MMTV-PyMT mouse-deficient in miR155. We demonstrate that global loss of miR155 resulted in blunted tumor growth through modulating the tumor microenvironment. Specifically, miR155-deficient mice had smaller and less invasive tumors, an increase in apoptosis and a decrease in proliferation, a reduction in tumor-associated macrophages, and the expression of genes associated with protumoral processes.

Macrophage tumor necrosis factor-alpha deletion does not protect against obesity-associated metabolic dysfunction.

The pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), has been suggested to be a key factor in the induction of obesity-associated metabolic dysfunction. However, the role that macrophage-derived TNF-α has on regulating metabolic perturbations in obesity is not completely understood. Therefore, we utilized the TNF-αFlox/Flox(F/F) , LyzMcre± mouse model to determine the impact that macrophage TNF-α deletion has on the development of high-fat diet (HFD)-induced obesity. At 10 weeks of age, male littermates were randomly assigned to 1 of 4 groups: TNF-αF/F low-fat diet (TNF-αF/F LFD), TNF-αF/F,LyzMCre LFD, TNF-αF/F HFD, or TNF-αF/F,LyzMCre HFD (n = 16-28/group) and were fed their respective diets for 18 weeks. Body weight was assessed throughout the course of the experiment. Body composition, hepatic lipid accumulation, and metabolic outcomes were also examined. A microarray gene expression experiment was performed from RNA isolated from epididymal adipose tissue of the HFD-fed groups (n = 10/group) and results were verified via qRT-PCR for all groups. Macrophage-derived TNF-α deletion significantly reduced adipose tissue TNF-α gene expression and circulating TNF-α and downregulated genes linked to the toll-like receptor (TLR) and NFκB signaling pathways. However, macrophage TNF-α deletion had no effect on hindering the development of obesity, hepatic lipid accumulation, or improving glucose metabolism or insulin sensitivity. In conclusion, macrophage-derived TNF-α is not a causative factor for the induction of obesity-associated metabolic dysfunction.

Repeated clodronate-liposome treatment results in neutrophilia and is not effective in limiting obesity-linked metabolic impairments.

Depletion of macrophages is thought to be a therapeutic option for obesity-induced inflammation and metabolic dysfunction. However, whether the therapeutic effect is a direct result of reduced macrophage-derived inflammation or secondary to decreases in fat mass is controversial, as macrophage depletion has been shown to disrupt energy homeostasis. This study was designed to determine if macrophage depletion via clodronate-liposome (CLD) treatment could serve as an effective intervention to reduce obesity-driven inflammatory and metabolic impairments independent of changes in energy intake. After 16 wk on a high-fat diet (HFD) or the AIN-76A control (low-fat) diet (LFD) ( n = 30/diet treatment), male C57BL/6J mice were assigned to a CLD- or PBS-liposome treatment ( n = 15/group) for 4 wk. Liposomes were administered biweekly via intraperitoneal injections (8 administrations in total). PBS-liposome-treated groups were pair-fed to their CLD-treated dietary counterparts. Metabolic function was assessed before and after liposome treatment. Adipose tissue, as well as the liver, was investigated for macrophage infiltration and the presence of inflammatory mediators. Additionally, a complete blood count was performed. CLD treatment reduced energy intake. When controlling for energy intake, CLD treatment was unable to regress metabolic dysfunction or nonalcoholic fatty liver disease and impaired adipose tissue insulin action. Moreover, repeated CLD treatment induced neutrophilia and anemia, increased adipose tissue mRNA expression of the proinflammatory cytokines IL-6 and IL-1ß, and augmented circulating IL-6 and monocyte chemoattractant protein-1 concentrations ( P < 0.05). This study suggests that repeated intraperitoneal administration of CLD to deplete macrophages attenuates obesity by limiting energy intake. Moreover, after controlling for the benefits of weight loss, the accompanying detrimental side effects limit regular CLD treatment as an effective therapeutic strategy.

Macrophage depletion using clodronate liposomes decreases tumorigenesis and alters gut microbiota in the AOM/DSS mouse model of colon cancer.

We examined the role of macrophages in inflammation associated with colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice ( n = 32) received a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulfate (DSS)-supplemented water in weeks 1, 4, and 7. Prior to the final DSS cycle ( week 7) and twice weekly until euthanasia, mice ( n = 16/group) received either 200 µl ip of clodronate-filled liposomes (CLD) or phosphate-buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected, and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ∼36% and specifically large (≥1 mm) tumors by ∼36% ( P < 0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFß, CCL17) in the colon tissue ( P < 0.05). Similarly, there was a decrease in STAT3 and p38 MAPK and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum ( P < 0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC. NEW & NOTEWORTHY We found that macrophage depletion during late-stage tumorigenesis is effective at reducing tumor growth. This was associated with a decrease in macrophage markers and chemokines in the colon tissue and a decrease in transcription factors that are linked to colorectal cancer. The macrophage-depleted group was found to have an increased abundance of Firmicutes, a phylum with documented anti-tumorigenic effects. Overall, these data support the development of therapeutic strategies to target macrophages in colorectal cancer.

MicroRNA-155 deletion promotes tumorigenesis in the azoxymethane-dextran sulfate sodium model of colon cancer.

Clinical studies have linked microRNA-155 (miR-155) expression in the tumor microenvironment to poor prognosis. However, whether miR-155 upregulation is predictive of a pro- or antitumorigenic response is unclear, as the limited preclinical data available remain controversial. We examined miR-155 expression in tumor tissue from colon cancer patients. Furthermore, we investigated the role of this microRNA in proliferation and apoptosis, inflammatory processes, immune cell populations, and transforming growth factor-ß/SMAD signaling in a chemically induced (azoxymethane-dextran sulfate sodium) mouse model of colitis-associated colon cancer. We found a higher expression of miR-155 in the tumor region than in nontumor colon tissue of patients with colon cancer. Deletion of miR-155 in mice resulted in a greater number of polyps/adenomas, an increased symptom severity score, a higher grade of epithelial dysplasia, and a decrease in survival. Surprisingly, these findings were associated with an increase in apoptosis in the normal mucosa, but there was no change in proliferation. The protumorigenic effects of miR-155 deletion do not appear to be driven solely by dysregulation of inflammation, as both genotypes had relatively similar levels of inflammatory mediators. The enhanced tumorigenic response in miR-155(-/-) mice was associated with alterations in macrophages and neutrophils, as markers for these populations were decreased and increased, respectively. Furthermore, we demonstrated a greater activation of the transforming growth factor-ß/SMAD pathway in miR-155(-/-) mice, which was correlated with the increased tumorigenesis. Given the multiple targets of miR-155, careful evaluation of its role in tumorigenesis is necessary prior to any consideration of its potential as a biomarker and/or therapeutic target in colon cancer.

High-fat diets rich in saturated fat protect against azoxymethane/dextran sulfate sodium-induced colon cancer.

High-fat-diet (HFD) consumption is associated with colon cancer risk. However, little is known about how the lipid composition of a HFD can influence prooncogenic processes. We examined the effects of three HFDs differing in the percentage of total calories from saturated fat (SF) (6, 12, and 24% of total caloric intake), but identical in total fat (40%), and a commercially available Western diet (26 and 41% saturated and total fat, respectively) on colon cancer development using the azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model. A second dose-response experiment was performed using diets supplemented with the saturated-fatty-acid (SFA)-rich coconut oil. In experiment 1, we found an inverse association between SF content and tumor burden. Furthermore, increased SF content was associated with reduced inflammation, increased apoptosis, and decreased proliferation. The second dose-response experiment was performed to test whether this effect may be attributed to the SF content of the diets. Consistent with the initial experiment, we found that high SF content was protective, at least in male mice; there was a decrease in mortality in mice consuming the highest concentration of SFAs. To explore a potential mechanism for these findings, we examined colonic mucin 2 (Muc2) protein content and found that the HFDs with the highest SF content had the greatest concentration of Muc2. Our data suggest that high dietary SF is protective in the AOM/DSS model of colon cancer, which may be due, at least in part, to the ability of SF to maintain intestinal barrier integrity through increased colonic Muc2.

Weight loss following diet-induced obesity does not alter colon tumorigenesis in the AOM mouse model.

Obesity presents a significant public health concern given its association with increased cancer incidence, unfavorable prognosis, and metastasis. However, there is very little literature on the effects of weight loss, following obesity, on risk for colon cancer or liver cancer. Therefore, we sought to study whether intentional weight loss through diet manipulation was capable of mitigating colon and liver cancer in mice. We fed mice with a high-fat diet (HFD) comprised of 47% carbohydrates, 40% fat, and 13% protein for 20 wk to mimic human obesity. Subsequently, azoxymethane (AOM) was used to promote colon and liver carcinogenesis. A subset of obese mice was then switched to a low-fat diet (LFD) containing 67.5% carbohydrate, 12.2% fat, and 20% protein to promote intentional weight loss. Body weight loss and excess fat reduction did not protect mice from colon cancer progression and liver dysplastic lesion in the AOM-chemical-cancer model even though these mice had improved blood glucose and leptin levels. Intentional weight loss in AOM-treated mice actually produced histological changes that resemble dysplastic alterations in the liver and presented a higher percentage of F4/80+CD206+ macrophages and activated T cells (CD4+CD69+) in the spleen and lymph nodes, respectively. In addition, the liver of AOM-treated mice exposed to a HFD during the entire period of the experiment exhibited a marked increase in proliferation and pNF-κB activation. Altogether, these data suggest that intentional weight loss following chemical-induced carcinogenesis does not affect colon tumorigenesis but may in fact negatively impact liver repair mechanisms.

Influence of high-fat diet on gut microbiota: a driving force for chronic disease risk.

PURPOSE OF REVIEW: This review will examine the recent scientific literature surrounding high-fat-diet (HFD)-induced alterations in gut microbiota and subsequent development of obesity and chronic disease risk. RECENT FINDINGS: Excessive consumption of HFDs has undoubtedly contributed to the obesity epidemic. The mechanisms responsible for this relationship are, however, likely to be more complex than the simple concept of energy balance. In fact, emerging literature has implicated HFD-induced alterations in gut microbiota in the obesity epidemic. HFD consumption generally leads to a decrease in Bacteroidetes and an increase in Firmicutes, alterations that have been associated with obesity and subsequent development of chronic diseases. Potential mechanisms for this effect include an improved capacity for energy harvest and storage, and enhanced gut permeability and inflammation. We highlight the most important recent advances linking HFD-induced dysbiosis to obesity, explore the possible mechanisms for this effect, examine the implications for disease development, and evaluate the possibility of therapeutic targeting of the gut microbiome to reduce obesity. SUMMARY: A better understanding of the mechanisms linking HFD to alterations in gut microbiota is necessary to allow for the regulation of dysbiosis and ensuing promotion of antiobesity effects.

Influence of Exercise on Inflammation in Cancer: Direct Effect or Innocent Bystander?

We propose the hypothesis that the benefits of exercise on inflammation in cancer are a result of a direct effect on inflammatory cytokines, including interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein 1, that are critical for cancer growth as well as a bystander effect of the established relationship between exercise and cancer.

Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice.

Although there are currently no approved treatments for cancer cachexia, there is an intensified interest in developing therapies because of the high mortality index associated with muscle wasting diseases. Successful treatment of the cachectic patient focuses on improving or maintaining body weight and musculoskeletal function. Nutraceutical compounds, including the natural phytochemical quercetin, are being examined as potential treatments because of their anti-inflammatory, antioxidant, and anticarcinogenic properties. The purpose of this study was to determine the effect of quercetin supplementation on the progression of cachexia in the adenomatous polyposis coli (Apc)(Min/+) mouse model of colorectal cancer. At 15 wk of age, C57BL/6 and male Apc(Min/+) mice were supplemented with 25 mg/kg of quercetin or vehicle solution mix of Tang juice and water (V) daily for 3 wk. Body weight, strength, neuromuscular performance, and fatigue were assessed before and after quercetin or V interventions. Indicators of metabolic dysfunction and inflammatory signaling were also assessed. During the treatment period, the relative decrease in body weight in the Apc(Min/+) mice gavaged with V (Apc(Min/+)V; -14% ± 2.3) was higher than in control mice gavaged with V (+0.6% ± 1.0), control mice gavaged with quercetin (-2% ± 1.0), and Apc(Min/+) mice gavaged with quercetin (Apc(Min/+)Q; -9% ± 1.3). At 18 wk of age, the loss of grip strength and muscle mass shown in Apc(Min/+)V mice was significantly attenuated (P < 0.05) in Apc(Min/+)Q mice. Furthermore, Apc(Min/+)V mice had an induction of plasma interleukin-6 and muscle signal transducer and activator of transcription 3 phosphorylation, which were significantly (P < 0.05) mitigated in Apc(Min/+)Q mice, despite having a similar tumor burden. Quercetin treatment did not improve treadmill run-time-to-fatigue, hyperglycemia, or hyperlipidemia in cachectic Apc(Min/+) mice. Overall, quercetin supplementation positively affected several aspects of cachexia progression in mice and warrants further exploration as a potential anticachectic therapeutic.

Obesity worsens mitochondrial quality control and does not protect against skeletal muscle wasting in murine cancer cachexia.

BACKGROUND: More than 650 million people are obese (BMI > 30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. METHODS: Male C57BL/6 mice were given a purified high fat or standard diet for 16 weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25 days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). RESULTS: Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P = 0.0001) but had reduced survival (33.3% vs. 6.67%, χ2  = 10.04, P = 0.0182). Obese LLC mice had reduced muscle weights (-24%, P < 0.0354) and mCSA (-16%, P = 0.0004) with similar activation of muscle p65 (P = 0.0337), and p38 (P = 0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P = 0.0072) consistent with reductions in volitional cage activity (-39%, P < 0.0001) and grip strength (-41%, P < 0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P = 0.0009; SS: -21%, P = 0.0101) and LLC (IMF: -40%, P = 0.0019; SS: -27%, P = 0.0383) mice. Obese LLC mice had increased pAMPK (T172; P = 0.0103) and reduced FIS1 (P = 0.0029) and DRP1 (P < 0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P = 0.0395; SS: 138%, P = 0.0174) in concert with an accumulation of p62 (P = 0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P = 0.0260; SS: 392%, P = 0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. CONCLUSIONS: Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.

A Skeletal Muscle-Mediated Anticontractile Response on Vascular Tone: Unraveling the Lactate-AMPK-NOS1 Pathway in Femoral Arteries.

The regulation of vascular tone by perivascular tissues is a complex interplay of various paracrine factors. Here, we investigate the anti-contractile effect of skeletal muscle surrounding the femoral and carotid arteries and its underlying mechanisms. Using male and female Wistar rats, we demonstrated that serotonin, phenylephrine, and U-46619 induced a concentration-dependent vasoconstrictor response in femoral artery rings. Interestingly, this response was diminished in the presence of surrounding femoral skeletal muscle, irrespective of sex. No anti-contractile effect was observed when the carotid artery was exposed to its surrounding skeletal muscle. The observed effect in the femoral artery persisted even in the absence of endothelium and when the muscle was detached from the artery. Furthermore, the skeletal muscle surrounding the femoral artery was able to promote an anti-contractile effect in three other vascular beds (basilar, mesenteric, and carotid arteries). Using inhibitors of lactate dehydrogenase and the 1/4 monocarboxylate transporter, we confirmed the involvement of lactate, as both inhibitors were able to abolish the anti-contractile effect. However, lactate did not directly promote vasodilation; rather, it exerted its effect by activating 5' AMP-activated protein kinase (AMPK) and neuronal nitric oxide synthase (NOS1) in the skeletal muscle. Accordingly, Nω-propyl L-arginine, a specific inhibitor of NOS1, prevented the anti-contractile effect, as well as lactate-induced phosphorylation of NOS1 at the stimulatory serine site (1417) in primary skeletal muscle cells. Phosphorylation of NOS1 was reduced in the presence of Bay-3827, a selective AMPK inhibitor. In conclusion, femoral artery-associated skeletal muscle is a potent paracrine and endocrine organ that influences vascular tone in both sexes. Mechanistically, the anti-contractile effect involves muscle fiber type and/or its anatomical location but not the type of artery or its related vascular endothelium. Finally, the femoral artery anti-contractile effect is mediated by the lactate-AMPK-phospho-NOS1Ser1417-NO signaling axis.

Influence of dietary saturated fat content on adiposity, macrophage behavior, inflammation, and metabolism: composition matters.

We examined the effects of three high-fat diets (HFD), differing in the percentage of total calories from saturated fat (SF) (6%, 12%, and 24%) but identical in total fat (40%), on body composition, macrophage behavior, inflammation, and metabolic dysfunction in mice. Diets were administered for 16 weeks. Body composition and metabolism [glucose, insulin, triglycerides, LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), total cholesterol (TC)] were examined monthly. Adipose tissue (AT) expression of marker genes for M1 and M2 macrophages and inflammatory mediators [Toll-like receptor (TLR)-2, TLR-4, MCP-1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, suppressor of cytokine signaling (SOCS)1, IFN-γ] was measured along with activation of nuclear factor kappa-B (NFκB), c-Jun N-terminal kinase (JNK), and p38- mitogen-activated protein kinase (MAPK). AT macrophage infiltration was examined using immunohistochemistry. Circulating MCP-1, IL-6, adiponectin, and leptin were also measured. SF content, independent of total fat, can profoundly affect adiposity, macrophage behavior, inflammation, and metabolic dysfunction. In general, the 12%-SF diet, most closely mimicking the standard American diet, led to the greatest adiposity, macrophage infiltration, and insulin resistance (IR), whereas the 6%-SF and 24%-SF diets produced lower levels of these variables, with the 24%-SF diet resulting in the least degree of IR and the highest TC/HDL-C ratio. Macrophage behavior, inflammation, and IR following HFD are heavily influenced by dietary SF content; however, these responses are not necessarily proportional to the SF percentage.

Linking inflammation to tumorigenesis in a mouse model of high-fat-diet-enhanced colon cancer.

Many observational epidemiologic studies suggest an association between high-fat-diet (HFD) and colon cancer risk. However, the lack of controlled experimental studies that examine this relationship and the mechanisms involved weaken the basis for inferring a causal relationship. Inflammation plays a role in colon cancer progression and HFDs have been reported to increase inflammation; however, the inflammatory effects of HFD in colon cancer have yet to be firmly established. We examined the effects of a novel HFD that closely mimics the standard American diet (12% and 40% of total caloric intake from saturated fat and total fat, respectively) on macrophage markers and inflammatory mediators in a mouse model of intestinal tumorigenesis and relate this to polyp characteristics as well as measures of adiposity. Male Apc(Min/+) mice (7-8/group) were fed a Control Diet (Con) or novel high-fat-diet (HFD) from 4 to 12weeks of age. Body weight and body composition were measured weekly and monthly, respectively. Intestinal tissue was analyzed for polyp burden (number and size). Gene expression of macrophage markers and inflammatory mediators were examined in the adipose tissue and polyps. The HFD increased the expression of macrophage markers and inflammatory mediators in the adipose tissue (F4/80, CD11c, TLR-4 and MCP-1) and tumor microenvironment (IL-12, MCP-1, IL-6 and TNF-α). As expected, the HFD increased body weight, body fat percent, fat mass and blood glucose (P<0.05), and was associated with an increase in the number of large polyps (P<0.05) but not total polyps. In summary, consumption of a HFD, similar in macronutrient composition to the standard American diet, altered the expression of macrophage phenotypic markers and inflammatory mediators in adipose tissue and intestinal polyps and this was associated with increased tumorigenesis.

How stable is your vivarium's temperature? Fluctuations in vivarium temperature significantly impact metabolism and behavior impeding scientific reproducibility.

OBJECTIVES: The purpose of this investigation was to examine the variability in vivarium temperature and the impact that this has on metabolic and behavioral outcomes in mice. METHODS: Daily vivarium temperature was monitored every day for a two-year period. Behavioral and metabolic phenotyping were assessed in male and female C57BL/6 (n = 71/sex) mice over the course of 2 years. RESULTS: Vivarium temperature was found to fluctuate on a monthly, daily, and even an hourly basis of approximately ±5ºC. A 5ºC change in temperature was found to result in daily changes in total energy expenditure (35% and 27%), resting energy expenditure (39% for both sexes), movement (51% and 37%), food consumption (35% and 29%), and sleep duration (15% and 13%) for female and male mice, respectively. CONCLUSIONS: Fluctuations in vivarium temperature can dramatically impact metabolic and behavioral outcomes, which impedes scientific reproducibility. This awareness and the guidelines we propose in this publication will hopefully help to enhance the reproducibility of pre-clinical animal research.

Skeletal Muscle Endogenous Estrogen Production Ameliorates the Metabolic Consequences of a High-Fat Diet in Male Mice.

AIMS: The role of skeletal muscle estrogen and its ability to mitigate the negative impact of a high-fat diet (HFD) on obesity-associated metabolic impairments is unknown. To address this, we developed a novel mouse model to determine the role of endogenous 17ß-estradiol (E2) production in males in skeletal muscle via inducible, skeletal muscle-specific aromatase overexpression (SkM-Arom↑). METHODS: Male SkM-Arom↑ mice and littermate controls were fed a HFD for 14 weeks prior to induction of SkM-Arom↑ for a period of 6.5 weeks. Glucose tolerance, insulin action, adipose tissue inflammation, and body composition were assessed. Indirect calorimetry and behavioral phenotyping experiments were performed using metabolic cages. Liquid chromatography mass spectrometry was used to determine circulating and tissue (skeletal muscle, hepatic, and adipose) E2 and testosterone concentrations. RESULTS: SkM-Arom↑ significantly increased E2 in skeletal muscle, circulation, the liver, and adipose tissue. SkM-Arom↑ ameliorated HFD-induced hyperglycemia, hyperinsulinemia, impaired glucose tolerance, adipose tissue inflammation, and reduced hepatic lipid accumulation while eliciting skeletal muscle hypertrophy. CONCLUSION: Enhanced skeletal muscle aromatase activity in male mice induces weight loss, improves metabolic and inflammatory outcomes and mitigates the negative effects of a HFD. Additionally, our data demonstrate for the first time skeletal muscle E2 has anabolic effects on the musculoskeletal system.

Platelet status in cancer cachexia progression in ApcMin/+ mice.

Cachexia, a complex wasting syndrome, significantly affects the quality of life and treatment options for cancer patients. Studies have reported a strong correlation between high platelet count and decreased survival in cachectic individuals. Therefore, this study aimed to investigate the immunopathogenesis of cancer cachexia using the ApcMin/+ mouse model of spontaneous colorectal cancer. The research focused on identifying cellular elements in the blood at different stages of cancer cachexia, assessing inflammatory markers and fibrogenic factors in the skeletal muscle, and studying the behavioral and metabolic phenotype of ApcMin/+ mice at the pre-cachectic and severely cachectic stages. Platelet measurements were also obtained from other animal models of cancer cachexia - Lewis Lung Carcinoma and Colon 26 adenocarcinoma. Our study revealed that platelet number is elevated prior to cachexia development in ApcMin/+ mice and can become activated during its progression. We also observed increased expression of TGFß2, TGFß3, and SMAD3 in the skeletal muscle of pre-cachectic ApcMin/+ mice. In severely cachectic mice, we observed an increase in Ly6g, CD206, and IL-10 mRNA. Meanwhile, IL-1ß gene expression was elevated in the pre-cachectic stage. Our behavioral and metabolic phenotyping results indicate that pre-cachectic ApcMin/+ mice exhibit decreased physical activity. Additionally, we found an increase in anemia at pre-cachectic and severely cachectic stages. These findings highlight the altered platelet status during early and late stages of cachexia and provide a basis for further investigation of platelets in the field of cancer cachexia.

A Novel Tissue-Specific Insight into Sex Steroid Fluctuations Throughout the Murine Estrous Cycle.

Serum sex steroid levels fluctuate throughout the reproductive cycle. However, the degree to which sex steroid tissue content mimics circulating content is unknown. Understanding the flux and physiological quantity of tissue steroid content is imperative for targeted hormonal therapy development. Utilizing a gold-standard ultrasensitive liquid chromatography-mass spectrometry (LC/MS) method we determined sex steroid (17ß-estradiol [E2], testosterone, androstenedione, and progesterone) fluctuations in serum and in 15 tissues throughout the murine estrous cycle (proestrus, estrus, and diestrus I) and in ovariectomized (OVX) mice. We observed dynamic fluctuations in serum and tissue steroid content throughout the estrous cycle with proestrus generally presenting the highest content of E2, testosterone, and androstenedione, and lowest content of progesterone. In general, the trend in circulating steroid content between the stages of the estrous cycle was mimicked in tissue. However, the absolute amounts of steroid levels when normalized to tissue weight were found to be significantly different between the tissues with the serum steroid quantity often being significantly lower than the tissue quantity. Additionally, we found that OVX mice generally displayed a depletion of all steroids in the various tissues assessed, except in the adrenal glands which were determined to be the main site of peripheral E2 production after ovary removal. This investigation provides a comprehensive analysis of steroid content throughout the estrous cycle in a multitude of tissues and serum. We believe this information will help serve as the basis for the development of physiologically relevant, tissue-specific hormonal therapies.