Combined metformin-salicylate treatment provides improved anti-tumor activity and enhanced radiotherapy response in prostate cancer; drug synergy at clinically relevant doses.

BACKGROUND: There is need for well-tolerated therapies for prostate cancer (PrCa) secondary prevention and to improve response to radiotherapy (RT). The anti-diabetic agent metformin (MET) and the aspirin metabolite salicylate (SAL) are shown to activate AMP-activated protein kinase (AMPK), suppress de novo lipogenesis (DNL), the mammalian target of rapamycin (mTOR) pathway and reduce PrCa proliferation in-vitro. The purpose of this study was to examine whether combined MET+SAL treatment could provide enhanced PrCa tumor suppression and improve response to RT. METHODS: Androgen-sensitive (22RV1) and resistant (PC3, DU-145) PrCa cells and PC3 xenografts were used to examine whether combined treatment with MET+SAL can provide improved anti-tumor activity compared to each agent alone in non-irradiated and irradiated PrCa cells and tumors. Mechanisms of action were investigated with analysis of signaling events, mitochondria respiration and DNL activity assays. RESULTS: We observed that PrCa cells are resistant to clinically relevant doses of MET. Combined MET + SAL treatment provides synergistic anti-proliferative activity at clinically relevant doses and enhances the anti-proliferative effects of RT. This was associated with suppression of oxygen consumption rate (OCR), activation of AMPK, suppression of acetyl-CoA carboxylase (ACC)-DNL and mTOR-p70s6k/4EBP1 and HIF1α pathways. MET + SAL reduced tumor growth in non-irradiated tumors and enhanced the effects of RT. CONCLUSION: MET+SAL treatment suppresses PrCa cell proliferation and tumor growth and enhances responses to RT at clinically relevant doses. Since MET and SAL are safe, widely-used and inexpensive agents, these data support the investigation of MET+SAL in PrCa clinical trials alone and in combination with RT.

Salicylate enhances the response of prostate cancer to radiotherapy.

BACKGROUND: Radiotherapy (RT) is a key therapeutic modality for prostate cancer (PrCa), but RT resistance necessitates dose-escalation, often causing bladder and rectal toxicity. Aspirin, a prodrug of salicylate (SAL), has been associated with improved RT response in clinical PrCa cases, but the potential mechanism mediating this effect is unknown. SAL activates the metabolic stress sensor AMP-activated protein kinase (AMPK), which inhibits de novo lipogenesis, and protein synthesis via inhibition of Acetyl-CoA Carboxylase (ACC), and the mammalian Target of Rapamycin (mTOR), respectively. RT also activates AMPK through a mechanism distinctly different from SAL. Therefore, combining these two therapies may have synergistic effects on suppressing PrCa. Here, we examined the potential of SAL to enhance the response of human PrCa cells and tumors to RT. METHODS: Androgen-insensitive (PC3) and -sensitive (LNCaP) PrCa cells were subjected to proliferation and clonogenic survival assays after treatment with clinically relevant doses of SAL and RT. Balb/c nude mice with PC3 xenografts were fed standard chow diet or chow diet supplemented with 2.5 g/kg salsalate (SAL pro-drug dimer) one week prior to a single dose of 0 or 10 Gy RT. Immunoblotting analysis of signaling events in the DNA repair and AMPK-mTOR pathways and lipogenesis were assessed in cells treated with SAL and RT. RESULTS: SAL inhibited proliferation and clonogenic survival in PrCa cells and enhanced the inhibition mediated by RT. Salsalate, added to diet, enhanced the anti-tumor effects of RT in PC3 tumor xenografts. RT activated genotoxic stress markers and the activity of mTOR pathway and AMPK and mediated inhibitory phosphorylation of ACC. Interestingly, SAL enhanced the effects of RT on AMPK and ACC but blocked markers of mTOR activation. CONCLUSIONS: Our results show that SAL can enhance RT responses in PrCa. Salsalate is a promising agent to investigate this concept in prospective clinical trials of PrCa in combination with RT.

High Intensity Interval Training Increases Natural Killer Cell Number and Function in Obese Breast Cancer-challenged Mice and Obese Women.

High intensity interval training (HIIT) boosts natural killer (NK) cell number and activity in normal weight breast cancer patients; however, whether this occurs in obese individuals is not well established. The goal of this study was to determine whether HIIT effectively boosts NK cells as a therapeutic strategy against breast cancer in an obese mouse model and in overweight/obese women. Diet induced female C57Bl/6 obese mice were assigned to undergo HIIT for four weeks or remain sedentary. Female participants were subjected to a six weeks HIIT protocol. HIIT mice acclimatized to treadmill running were subsequently injected with 5 × 105 polyoma middle T (MT) breast cancer cells intravenously. NK cell number and activation were monitored using flow cytometry, and tumor burden or lipid content evaluated from histological lung and liver tissues, respectively. In both mice and humans, circulating NK cell number and activation (CD3-NK1.1+CD27+ and CD3-CD56+, respectively) markedly increased immediately after HIIT. HIIT obese mice had reduced lung tumor burden compared to controls following MT challenge, and had diminished hepatic lipid deposition despite minimal body weight loss. Our findings demonstrate that HIIT can benefit obese individuals by enhancing NK cell number and activity, reducing tumor burden, and enhancing metabolic health.

The diabetes medication Canagliflozin reduces cancer cell proliferation by inhibiting mitochondrial complex-I supported respiration.

OBJECTIVE: The sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate that SGLT2 inhibitors may inhibit the growth of some cancer cells but the mechanism(s) remain unclear. METHODS: Cellular proliferation and clonogenic survival were used to assess the sensitivity of prostate and lung cancer cell growth to the SGLT2 inhibitors. Oxygen consumption, extracellular acidification rate, cellular ATP, glucose uptake, lipogenesis, and phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase, and the p70S6 kinase were assessed. Overexpression of a protein that maintains complex-I supported mitochondrial respiration (NDI1) was used to establish the importance of this pathway for mediating the anti-proliferative effects of Canagliflozin. RESULTS: Clinically achievable concentrations of Canagliflozin, but not Dapagliflozin, inhibit cellular proliferation and clonogenic survival of prostate and lung cancer cells alone and in combination with ionizing radiation and the chemotherapy Docetaxel. Canagliflozin reduced glucose uptake, mitochondrial complex-I supported respiration, ATP, and lipogenesis while increasing the activating phosphorylation of AMPK. The overexpression of NDI1 blocked the anti-proliferative effects of Canagliflozin indicating reductions in mitochondrial respiration are critical for anti-proliferative actions. CONCLUSION: These data indicate that like the biguanide metformin, Canagliflozin not only lowers blood glucose but also inhibits complex-I supported respiration and cellular proliferation in prostate and lung cancer cells. These observations support the initiation of studies evaluating the clinical efficacy of Canagliflozin on limiting tumorigenesis in pre-clinical animal models as well epidemiological studies on cancer incidence relative to other glucose lowering therapies in clinical populations.

Treatment of nonalcoholic fatty liver disease: role of AMPK.

Nonalcoholic fatty liver disease (NAFLD) is a growing worldwide epidemic and an important risk factor for the development of insulin resistance, type 2 diabetes, nonalcoholic steatohepatitis (NASH), and hepatic cellular carcinoma (HCC). Despite the prevalence of NAFLD, lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease. Over the last decade, numerous experimental compounds have been shown to improve NAFLD in preclinical animal models, and many of these therapeutics have been shown to increase the activity of the cellular energy sensor AMP-activated protein kinase (AMPK). Because AMPK activity is reduced by inflammation, obesity, and diabetes, increasing AMPK activity has been viewed as a viable therapeutic strategy to improve NAFLD. In this review, we propose three primary mechanisms by which AMPK activation may improve NAFLD. In addition, we examine the mechanisms by which AMPK is activated. Finally, we identify 27 studies that have used AMPK activators to reduce NAFLD. Future considerations for studies examining the relationship between AMPK and NAFLD are highlighted.

Salsalate (Salicylate) Uncouples Mitochondria, Improves Glucose Homeostasis, and Reduces Liver Lipids Independent of AMPK-ß1.

Salsalate is a prodrug of salicylate that lowers blood glucose in patients with type 2 diabetes (T2D) and reduces nonalcoholic fatty liver disease (NAFLD) in animal models; however, the mechanism mediating these effects is unclear. Salicylate directly activates AMPK via the ß1 subunit, but whether salsalate requires AMPK-ß1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-ß1-knockout (AMPK-ß1KO) mice were treated with a salsalate dose resulting in clinically relevant serum salicylate concentrations (∼1 mmol/L). Salsalate treatment increased VO2, lowered fasting glucose, improved glucose tolerance, and led to an ∼55% reduction in liver lipid content. These effects were observed in both WT and AMPK-ß1KO mice. To explain these AMPK-independent effects, we found that salicylate increases oligomycin-insensitive respiration (state 4o) and directly increases mitochondrial proton conductance at clinical concentrations. This uncoupling effect is tightly correlated with the suppression of de novo lipogenesis. Salicylate is also able to stimulate brown adipose tissue respiration independent of uncoupling protein 1. These data indicate that the primary mechanism by which salsalate improves glucose homeostasis and NAFLD is via salicylate-driven mitochondrial uncoupling.

High-intensity exercise training increases the diversity and metabolic capacity of the mouse distal gut microbiota during diet-induced obesity.

Diet and exercise underpin the risk of obesity-related metabolic disease. Diet alters the gut microbiota, which contributes to aspects of metabolic disease during obesity. Repeated exercise provides metabolic benefits during obesity. We assessed whether exercise could oppose changes in the taxonomic and predicted metagenomic characteristics of the gut microbiota during diet-induced obesity. We hypothesized that high-intensity interval training (HIIT) would counteract high-fat diet (HFD)-induced changes in the microbiota without altering obesity in mice. Compared with chow-fed mice, an obesity-causing HFD decreased the Bacteroidetes-to-Firmicutes ratio and decreased the genetic capacity in the fecal microbiota for metabolic pathways such as the tricarboxylic acid (TCA) cycle. After HFD-induced obesity was established, a subset of mice were HIIT for 6 wk, which increased host aerobic capacity but did not alter body or adipose tissue mass. The effects of exercise training on the microbiota were gut segment dependent and more extensive in the distal gut. HIIT increased the alpha diversity and Bacteroidetes/Firmicutes ratio of the distal gut and fecal microbiota during diet-induced obesity. Exercise training increased the predicted genetic capacity related to the TCA cycle among other aspects of metabolism. Strikingly, the same microbial metabolism indexes that were increased by exercise were all decreased in HFD-fed vs. chow diet-fed mice. Therefore, exercise training directly opposed some of the obesity-related changes in gut microbiota, including lower metagenomic indexes of metabolism. Some host and microbial pathways appeared similarly affected by exercise. These exercise- and diet-induced microbiota interactions can be captured in feces.

The AMPK activator R419 improves exercise capacity and skeletal muscle insulin sensitivity in obese mice.

OBJECTIVE: Skeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects. METHODS: Glucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK ß1ß2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation. RESULTS: There was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice. CONCLUSIONS: Treatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity.

Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity.

Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK ß1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK ß1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.

High intensity interval training improves liver and adipose tissue insulin sensitivity.

OBJECTIVE: Endurance exercise training reduces insulin resistance, adipose tissue inflammation and non-alcoholic fatty liver disease (NAFLD), an effect often associated with modest weight loss. Recent studies have indicated that high-intensity interval training (HIIT) lowers blood glucose in individuals with type 2 diabetes independently of weight loss; however, the organs affected and mechanisms mediating the glucose lowering effects are not known. Intense exercise increases phosphorylation and inhibition of acetyl-CoA carboxylase (ACC) by AMP-activated protein kinase (AMPK) in muscle, adipose tissue and liver. AMPK and ACC are key enzymes regulating fatty acid metabolism, liver fat content, adipose tissue inflammation and insulin sensitivity but the importance of this pathway in regulating insulin sensitivity with HIIT is unknown. METHODS: In the current study, the effects of 6 weeks of HIIT were examined using obese mice with serine-alanine knock-in mutations on the AMPK phosphorylation sites of ACC1 and ACC2 (AccDKI) or wild-type (WT) controls. RESULTS: HIIT lowered blood glucose and increased exercise capacity, food intake, basal activity levels, carbohydrate oxidation and liver and adipose tissue insulin sensitivity in HFD-fed WT and AccDKI mice. These changes occurred independently of weight loss or reductions in adiposity, inflammation and liver lipid content. CONCLUSIONS: These data indicate that HIIT lowers blood glucose levels by improving adipose and liver insulin sensitivity independently of changes in adiposity, adipose tissue inflammation, liver lipid content or AMPK phosphorylation of ACC.

The role of AMPK in controlling metabolism and mitochondrial biogenesis during exercise.

Insulin resistance is associated with defects in skeletal muscle fatty acid (FA) metabolism that contribute to the development of type 2 diabetes. Endurance exercise increases FA and glucose metabolism, muscle mitochondrial content and insulin sensitivity. In skeletal muscle, basal rates of FA oxidation are dependent on AMP-activated protein kinase (AMPK) phosphorylation of acetyl-CoA carboxylase 2, the rate-limiting enzyme controlling the production of the metabolic intermediate malonyl-CoA. Likewise, AMPK is essential for maintaining muscle mitochondrial content in untrained mice; effects that may be mediated through regulation of the peroxisome proliferator-activated receptor Î³ co-activator-1α. However, the importance of AMPK in regulating glucose and FA uptake, FA oxidation and mitochondrial biogenesis during and following endurance exercise training is not fully understood. A better understanding of the mechanisms by which endurance exercise regulates substrate utilization and mitochondrial biogenesis may lead to improved therapeutic and preventative strategies for the treatment of insulin resistance and type 2 diabetes.

Reduced skeletal muscle AMPK and mitochondrial markers do not promote age-induced insulin resistance.

In both rodents and humans, aging-associated reductions in skeletal muscle AMP-activated protein kinase (AMPK) activity and mitochondrial function have been linked to the development of skeletal muscle insulin resistance. However, whether reductions in skeletal muscle AMPK and mitochondrial capacity actually precipitate the development of aging-induced insulin resistance is not known. Mice lacking both isoforms of the AMPK ß-subunit in skeletal muscle (AMPK-MKO) have no detectable AMPK activity and are characterized by large reductions in exercise capacity, mitochondrial content, and contraction-stimulated glucose uptake making them an ideal model to determine whether reductions in AMPK and mitochondrial content promote the development of aging-induced insulin resistance. In the current study we find that a lack of skeletal muscle AMPK results in a life-long reduction in mitochondrial activity but does not affect body mass, body composition, glucose tolerance, or insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp in mice of old age (18 mo). These data demonstrate that reductions in skeletal muscle AMPK and mitochondrial activity do not cause the development of age-induced insulin resistance.

Endurance interval training in obese mice reduces muscle inflammation and macrophage content independently of weight loss.

Obesity is associated with chronic low-grade inflammation that involves infiltration of macrophages into metabolic organs such as skeletal muscle. Exercise enhances skeletal muscle insulin sensitivity independently of weight loss; but its role in regulating muscle inflammation is not fully understood. We hypothesized that exercise training would inhibit skeletal muscle inflammation and alter macrophage infiltration into muscle independently of weight loss. Wild type C57BL/6 male mice were fed a chow diet or a high-fat diet (HFD, 45% calories fat) for 6 weeks. Then, mice maintained on the HFD either remained sedentary (HFD Sed) or exercised (HFD Ex) on a treadmill for another 6 weeks. The exercise training protocol involved conducting intervals of 2 min in duration followed by 2 min of rest for 60 min thrice weekly. Chow-fed control mice remained sedentary for the entire 12 weeks. Muscle cytokine and macrophage gene expression analysis were conducted using qRT-PCR, and muscle macrophage content was also measured using immunohistochemistry. Muscle cytokine protein content was quantified using a cytokine array. The HFD increased adiposity and weight gain compared to chow-fed controls. HFD Sed and HFD Ex mice had similar body mass as well as total and visceral adiposity. However, despite similar adiposity, exercise reduced inflammation and muscle macrophage infiltration. We conclude that Endurance exercise training modulates the immune-metabolic crosstalk in obesity independently of weight loss, and may have potential benefits in reducing obesity-related muscle inflammation.

Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin.

The obesity epidemic has led to an increased incidence of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP-activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed type 2 diabetes drug metformin. Ampk phosphorylates mouse acetyl-CoA carboxylase 1 (Acc1; refs. 3,4) at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl-CoA to malonyl-CoA. The latter metabolite is a precursor in fatty acid synthesis and an allosteric inhibitor of fatty acid transport into mitochondria for oxidation. To test the physiological impact of these phosphorylation events, we generated mice with alanine knock-in mutations in both Acc1 (at Ser79) and Acc2 (at Ser212) (Acc double knock-in, AccDKI). Compared to wild-type mice, these mice have elevated lipogenesis and lower fatty acid oxidation, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Notably, AccDKI mice made obese by high-fat feeding are refractory to the lipid-lowering and insulin-sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism and, in the setting of obesity, for metformin-induced improvements in insulin action.

Effects of prolonged treatment with memantine in the MRL model of CNS lupus.

OBJECTIVES: Neuropsychiatric manifestations and brain atrophy of unknown etiology are common and severe complications of systemic lupus erythematosus (SLE). An autoantibody that binds to N-methyl-D-aspartate (NMDA) receptor NR2 has been proposed as a key factor in the etiology of central nervous system (CNS) SLE. This hypothesis was supported by evidence suggesting memantine (MEM), an uncompetitive NMDA receptor antagonist, prevents behavioral dysfunction and brain pathology in healthy mice immunized with a peptide similar to an epitope on the NR2 receptor. Given that SLE is a chronic condition, we presently examine the effects of MEM in MRL/lpr mice, which develop behavioral deficits alongside SLE-like disease. METHODS: A broad behavioral battery and 7-Tesla MRI were used to examine whether prolonged treatment with MEM (~25 mg/kg b.w. in drinking water) prevents CNS involvement in this spontaneous model of SLE. RESULTS: Although MEM increased novel object exploration in MRL/lpr mice, it did not show other beneficial, substrain-specific effects. Conversely, MEM was detrimental to spontaneous activity in control MRL +/+ mice and had a negative effect on body mass gain. Similarly, MRI revealed comparable increases in the volume of periventricular structures in MEM-treated groups. CONCLUSIONS: Sustained exposure to MEM affects body growth, brain morphology, and behavior primarily by pharmacological, and not autoimmunity-dependant mechanisms. Substrain-specific improvement in exploratory behavior of MEM-treated MRL/lpr mice may indicate that the NMDA system is merely a constituent of a complex pathogenenic cascade. However, it was evident that chronic administration of MEM is unable to completely prevent the development of a CNS SLE-like syndrome.

Hematopoietic AMPK ß1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity.

Individuals who are obese are frequently insulin resistant, putting them at increased risk of developing type 2 diabetes and its associated adverse health conditions. The accumulation in adipose tissue of macrophages in an inflammatory state is a hallmark of obesity-induced insulin resistance. Here, we reveal a role for AMPK ß1 in protecting macrophages from inflammation under high lipid exposure. Genetic deletion of the AMPK ß1 subunit in mice (referred to herein as ß1(-/-) mice) reduced macrophage AMPK activity, acetyl-CoA carboxylase phosphorylation, and mitochondrial content, resulting in reduced rates of fatty acid oxidation. ß1(-/-) macrophages displayed increased levels of diacylglycerol and markers of inflammation, effects that were reproduced in WT macrophages by inhibiting fatty acid oxidation and, conversely, prevented by pharmacological activation of AMPK ß1-containing complexes. The effect of AMPK ß1 loss in macrophages was tested in vivo by transplantation of bone marrow from WT or ß1(-/-) mice into WT recipients. When challenged with a high-fat diet, mice that received ß1(-/-) bone marrow displayed enhanced adipose tissue macrophage inflammation and liver insulin resistance compared with animals that received WT bone marrow. Thus, activation of AMPK ß1 and increasing fatty acid oxidation in macrophages may represent a new therapeutic approach for the treatment of insulin resistance.