Insulin in combination with pioglitazone prevents advanced cachexia in 256-Walker tumor-bearing rats: effect is greater than treatment alone and is associated with improved insulin sensitivity.

BACKGROUND: Insulin (INS) resistance and hypoinsulinemia commonly observed in cancer-carrying, can contribute to cachexia. However, the effects of INS and INS sensitizers, such as pioglitazone (PIO), particularly when used in combination therapy, on cancer cachexia have not been evaluated sufficiently. We investigated the effects of INS and PIO, at various doses, either isolated or combined, on cachexia in Walker-256 tumor-bearing rats (TB rats). METHODS: INS or INS + PIO were administered in TB rats, for 6 or 12 days, starting from the day of tumor cells inoculation. RESULTS: INS at 18 or 27 U/kg (12-days treatment), but not 9 U/kg, reduced fat loss and slightly prevented weight loss. However, INS 18 U/kg + PIO 5, 10, 20, or 40 mg/kg (6 or 12-day treatment) reduced fat loss and markedly prevented weight loss but did not affect muscle wasting. While TB rats lost weight (37.9% in 12 days), TB rats treated with INS 18 U/kg + PIO 5 mg/kg showed pronounced weight gain (73.7%), which was greater than the sum (synergism) of the weight gains promoted by isolated treatments with INS 18 U/kg (14.7%) or PIO 5 mg/kg (13.1%). The beneficial effect of the INS 18 U/kg + PIO 5 mg/kg on weight loss was associated with improved INS sensitivity, as indicated by the higher blood glucose clearance constant (kITT), decreased levels of free fatty acids and triacylglycerols (INS resistance-inducing factors) in the blood, and increased expression of p-Akt (INS signaling pathway protein) in adipose tissue. CONCLUSIONS: The combined treatment with INS 18 U/kg + PIO 5 mg/kg was more effective in preventing advanced cachexia in TB rats than each treatment alone, emerging as the best approach, considering the lower dosage and higher efficacy. This combination completely preserved adipose mass and markedly reduced weight loss through a synergistic mechanism linked to improved insulin sensitivity. These findings provide new insights into the importance of drug combinations in effectively combating fat loss in advanced cachexia.

Gluconeogenesis is reduced from alanine, lactate and pyruvate, but maintained from glycerol, in liver perfusion of rats with early and late sepsis.

Sepsis induces several metabolic abnormalities, including hypoglycaemia in the most advanced stage of the disease, a risk factor for complications and death. Although hypoglycaemia can be caused by inhibition of hepatic gluconeogenesis, decreased and increased gluconeogenesis were reported in sepsis. Furthermore, gluconeogenesis from glycerol was not yet evaluated in this disease. The main purpose of this study was to investigate the gluconeogenesis from alanine, lactate, pyruvate and glycerol in rats with early (8 hours) and late (18 hours) sepsis. Parameters related to the characterization of sepsis were also evaluated. Sepsis was induced by cecal ligation and puncture and gluconeogenesis was assessed in liver perfusion. Rats with early and late sepsis showed increased lactataemia, depletion of liver glycogen and peripheral insulin resistance, characterizing the establishment of sepsis. Rats with early and late sepsis showed decreased gluconeogenesis from alanine, lactate and pyruvate. Interestingly, gluconeogenesis from glycerol, a precursor that enters in the pathway at a later step, subsequent to the entry of alanine, lactate and pyruvate, was maintained in rats with early and late sepsis. In conclusion, gluconeogenesis is decreased from alanine, lactate and pyruvate, but maintained from glycerol, in liver perfusion of rats with early and late sepsis. SIGNIFICANCE OF THE STUDY: The maintenance of gluconeogenesis from glycerol, but not from alanine, lactate and pyruvate, together with the liver glycogen depletion, points the glycerol as an important precursor for the maintenance of glycaemic homeostasis in sepsis. The findings open the possibility of further investigation on the administration of glycerol in the treatment of hypoglycaemia associated with more advanced sepsis.

Insulin secretion decline in Walker-256 tumor-bearing rats is early, follows the course of cachexia, and is not improved by lixisenatide.

Lixisenatide, a glucagon-like peptide-1 receptor agonist, is used to stimulate insulin secretion in patients with type 2 diabetes mellitus. However, its effect on insulin secretion in cancer patients, particularly during the cachexia course, has not yet been evaluated. The purpose of this study was to investigate the lixisenatide effect on INS secretion decline during the cachexia course (2, 6, and 12 days of tumor) in pancreatic islets isolated from Walker-256 tumor-bearing rats. Pancreatic islets of healthy and tumor-bearing rats were incubated in the presence or absence of lixisenatide (10 nM). Tumor-bearing rats showed reduction of body weight and fat and muscle mass, characterizing the development of cachexia, as well as reduction of insulinemia and INS secretion stimulated by glucose (5.6, 8.3, 11.1, 16.7, and 20 mM) on days 2, 6, and/or 12 of tumor. Lixisenatide increased the 16.7 mM glucose-stimulated insulin secretion, but not by 5.6 mM glucose, in the islets of healthy rats, without changing the insulin intracellular content. However, lixisenatide did not prevent the decreased 16.7 mM glucose-stimulated insulin secretion in the pancreatic islets of rats with 2, 6, and 12 days of tumor and neither the decreased insulin intracellular content of rats with 12 days of tumor. In consistency, in vivo treatment with lixisenatide (50 µg kg-1, SC, once daily, for 6 days) visually increased insulinemia of healthy fasted rats, but did not prevent hypoinsulinemia of tumor-bearing rats. In conclusion, Walker-256 tumor-bearing rats showed early decline (2 days of tumor) of insulin secretion, which followed the cachexia course (6 and 12 days of tumor) and was not improved by lixisenatide, evidencing that this insulin secretagogue, used to treat type 2 diabetes, does not have beneficial effect in cancer bearing-rats.

Akt activation by insulin treatment attenuates cachexia in Walker-256 tumor-bearing rats.

Cancer-bearing often exhibits hypoinsulinemia, insulin (INS) resistance and glutamine depletion associated with cachexia. However, INS and glutamine effects on cachexia metabolic abnormalities, particularly on tumor-affected proteins related to INS resistance, are poorly known. The main purpose of this study was to investigate the effects of INS and glutamine dipeptide (GDP) treatments on phospho-protein kinase B (p-Akt), and phospho-hormone sensitive lipase (p-HSL) in Walker-256 tumor-bearing rats. INS (NPH, 40 UI/kg, subcutaneous), GDP (1.5 g/kg, oral), INS+GDP or vehicle (control rats) were administered for 13 days, once a day, starting at the day of inoculation of tumor cells. The experiments were performed 4 hours after the last treatment to evaluate acute effects of INS and GDP, besides the chronic effects. INS and/or INS+GDP treatments, which markedly increased the insulinemia, increased the p-Akt: total Akt ratio and prevented the increased p-HSLSer552 : total HSL ratio in the retroperitoneal fat of tumor-bearing rats, without changing the INS resistance and increased expression of factor tumor necrosis-α (TNF-α) in this tissue. INS and INS+GDP also increased the p-Akt: total Akt ratio, whereas GDP and INS+GDP increased the GLUT4 glucose transporter gene expression, in the gastrocnemius muscle of the tumor-bearing rats. Accordingly, treatments with INS and INS+GDP markedly reduced glycemia, increased retroperitoneal fat and attenuated the body mass loss of tumor-bearing rats. In conclusion, hyperinsulinemia induced by high-dose INS treatments increased Akt phosphorylation and prevented increased p-HSLSer552 : total HSL ratio, overlapping INS resistance. These effects are consistent with increased fat mass gain and weight loss (cachexia) attenuation of tumor-bearing rats, evidencing that Akt activation is a potential strategy to prevent loss of fat mass in cancer cachexia.

Moderate exercise training since adolescence reduces Walker 256 tumour growth in adult rats.

KEY POINTS: Cancer growth, cell proliferation and cachexia index can be attenuated by the beneficial programming effect of moderate exercise training, especially if it begins in adolescence. Walker 256 tumour-bearing rats who started exercise training during adolescence did not revert the basal low glycaemia and insulinaemia observed before tumour cell inoculation. The moderate exercise training improved glucose tolerance and peripheral insulin sensitivity only in rats exercised early in adolescence. The chronic effects of our exercise protocol are be beneficial to prevent cancer cachexia and hold clear potential as a nonpharmacological therapy of insulin sensitization. ABSTRACT: We tested the hypothesis that moderate exercise training, performed early, starting during adolescence or later in life during adulthood, can inhibit tumour cell growth as a result of changes in biometric and metabolic markers. Male rats that were 30 and 70 days old performed a treadmill running protocol over 8 weeks for 3 days week-1 , 44 min day-1 and at 55-65% V̇O2max . After the end of training, a batch of rats was inoculated with Walker 256 carcinoma cells. At 15 days after carcinoma cell inoculation, the tumour was weighed and certain metabolic parameters were evaluated. The data demonstrated that physical performance was better in rats that started exercise training during adolescence according to the final workload and V̇O2max . Early or later moderate exercise training decreased the cachexia index, cell proliferation and tumour growth; however, the effects were more pronounced in rats that exercised during adolescence. Low glycaemia, insulinaemia and tissue insulin sensitivity was not reverted in Walker 256 tumour-bearing rats who trained during adolescence. Cancer growth can be attenuated by the beneficial programming effect of moderate exercise training, especially if it begins during adolescence. In addition, improvement in glucose-insulin homeostasis might be involved in this process.

Infusion of high concentration of lactate in perfused liver, simulating in vivo hyperlactatemia, prevents the reduction of gluconeogenesis in Walker-256 tumor-bearing rats.

Gluconeogenesis (GN) is increased in patients with cancer cachexia, but is reduced in liver perfusion of Walker-256 tumor-bearing cachectic rats (TB rats). The causes of these differences are unknown. We investigated the influence of circulating concentrations of lactate (NADH generator) and NADH on GN in perfused livers of TB rats. Lactate, at concentrations similar to those found on days 5 (3.0 mM), 8 (5.5 mM), and 12 (8.0 mM) of the tumor, prevented the reduction of GN from 2.0 mM lactate (lactatemia of healthy rat) in TB rats. NADH, 50 or 75 µM, but not 25 µM, increased GN from 2.0 mM lactate in TB rats to higher values than healthy rats. High concentrations of pyruvate (no NADH generator, 5.0 and 8.0 mM) did not prevent the reduction of GN from 2.0 mM pyruvate in TB rats. However, 50 or 75 µM NADH, but not 25 µM, increased GN from 2.0 mM pyruvate in TB rats to similar or higher values than healthy rats. High concentration of glutamine (NADH generator, 2.5 mM) or 50 µM NADH prevented the reduction of GN from 1 mM glutamine in TB rats. Intraperitoneal administration of pyruvate (1.0 mg/kg) or glutamine (0.5 mg/kg) similarly increased the glycemia of healthy and TB rats. In conclusion, high lactate concentration, similar to hyperlactatemia, prevented the reduction of GN in perfused livers of TB rats, an effect probably caused by the increased redox potential (NADH/NAD+ ). Thus, the decreased GN in livers from TB rats is due, at least in part, to the absence of simulation of in vivo hyperlactatemia in liver perfusion studies.

Aerobic Exercise Training Attenuates Tumor Growth and Reduces Insulin Secretion in Walker 256 Tumor-Bearing Rats.

Aerobic exercise training can improve insulin sensitivity in many tissues; however, the relationship among exercise, insulin, and cancer cell growth is unclear. We tested the hypothesis that aerobic exercise training begun during adolescence can attenuate Walker 256 tumor growth in adult rats and alter insulin secretion. Thirty-day-old male Wistar rats engaged in treadmill running for 8 weeks, 3 days/week, 44 min/day, at 55-65% VO2max until they were 90 days old (TC, Trained Control). An equivalently aged group was kept inactive during the same period (SC, Sedentary Control). Then, half the animals of the SC and TC groups were reserved as the control condition and the other half were inoculated with Walker 256 cancer cells, yielding two additional groups (Sedentary Walker and Trained Walker). Zero mortalities were observed in tumor-bearing rats. Body weight (BW), food intake, plasma glucose, insulin levels, and peripheral insulin sensitivity were analyzed before and after tumor cell inoculation. We also evaluated tumor growth, metastasis and cachexia. Isolated pancreatic islets secretory activity was analyzed. In addition, we evaluated mechanic sensibility. Our results showed improved physical performance according to the final workload and VO2max and reduced BW in trained rats at the end of the running protocol. Chronic adaptation to the aerobic exercise training decreased tumor weight, cachexia and metastasis and were associated with low glucose and insulin levels and high insulin sensitivity before and after tumor cell inoculation. Aerobic exercise started at young age also reduced pancreatic islet insulin content and insulin secretion in response to a glucose stimulus, without impairing islet morphology in trained rats. Walker 256 tumor-bearing sedentary rats also presented reduced pancreatic islet insulin content, without changing insulin secretion through isolated pancreatic islets. The mechanical sensitivity test indicated that aerobic exercise training did not cause injury or trigger inflammatory processes prior to tumor cell inoculation. Taken together, the current study suggests that aerobic exercise training applied during adolescence may mitigate tumor growth and related disorders in Walker 256 tumor-bearing adult rats. Improved insulin sensibility, lower glucose and insulin levels and/or reduced insulin secretion stimulated by glucose may be implicated in this tumor attenuation.

Effects of metformin on insulin resistance and metabolic disorders in tumor-bearing rats with advanced cachexia.

Metformin (MET) is widely used in the correction of insulin (INS) resistance and metabolic abnormalities in type 2 diabetes. However, its effect on INS resistance and metabolic disorders associated with cancer cachexia is not established. We investigated the MET effects, isolated or associated with INS, on INS resistance and metabolic changes induced by Walker-256 tumor in rats with advanced cachexia. MET (500 mg·kg-1, oral) and MET + INS (1.0 IU·kg-1, s.c.) were administered for 12 days, starting on the day of tumor cell inoculation. Tumor-bearing rats showed adipose and muscle mass wasting, body mass loss, anorexia, decreased Akt phosphorylation in retroperitoneal and mesenteric adipose tissue, peripheral INS resistance, hypoinsulinemia, reduced INS content and secretion from pancreatic islets, and also inhibition of glycolysis, gluconeogenesis, and glycogenolysis in liver. MET and MET + INS treatments did not prevent these changes. It can be concluded that treatments with MET and MET + INS did not prevent the adipose and muscle mass wasting and body mass loss of tumor-bearing rats possibly by not improving INS resistance. Therefore, MET, used for the treatment of INS resistance in type 2 diabetes, is not effective in improving INS resistance in the advanced stage of cancer cachexia, evidencing that the drug does not have the same beneficial effect in these 2 diseases.

Chronic Glibenclamide Treatment Attenuates Walker-256 Tumour Growth in Prediabetic Obese Rats.

BACKGROUND/AIMS: The sulphonylurea glibenclamide (Gli) is widely used in the treatment of type 2 diabetes. In addition to its antidiabetic effects, low incidences of certain types of cancer have been observed in Gli-treated diabetic patients. However, the mechanisms underlying this observation remain unclear. The aim of the present work was to evaluate whether obese adult rats that were chronically treated with an antidiabetic drug, glibenclamide, exhibit resistance to rodent breast carcinoma growth. METHODS: Neonatal rats were treated with monosodium L-glutamate (MSG) to induce prediabetes. Control and MSG groups were treated with Gli (2 mg/kg body weight/day) from weaning to 100 days old. After Gli treatment, the control and MSG rats were grafted with Walker-256 tumour cells. After 14 days, grafted rats were euthanized, and tumour weight as well as glucose homeostasis were evaluated. RESULTS: Treatment with Gli normalized tissue insulin sensitivity and glucose tolerance, suppressed fasting hyperinsulinaemia, reduced fat tissue accretion in MSG rats, and attenuated tumour growth by 27% in control and MSG rats. CONCLUSIONS: Gli treatment also resulted in a large reduction in the number of PCNA-positive tumour cells. Although treatment did improve the metabolism of pre-diabetic MSG-rats, tumour growth inhibition may be a more direct effect of glibenclamide.

Insulin, not glutamine dipeptide, reduces lipases expression and prevents fat wasting and weight loss in Walker 256 tumor-bearing rats.

Cachexia is the main cause of mortality in advanced cancer patients. We investigated the effects of insulin (INS) and glutamine dipeptide (GDP), isolated or associated, on cachexia and metabolic changes induced by Walker 256 tumor in rats. INS (NPH, 40 UI/kg, sc) or GDP (1.5g/kg, oral gavage) was once-daily administered during 11 days after tumor cell inoculation. GDP, INS or INS+GDP treatments did not influence the tumor growth. However, INS and INS+GDP prevented retroperitoneal fat wasting and body weight loss of tumor-bearing rats. In consistency, INS and INS+GDP prevented the increased expression of triacylglycerol lipase (ATGL) and hormone sensitive lipase (HSL), without changing the expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in the retroperitoneal adipose tissue of tumor-bearing rats. INS and INS+GDP also prevented anorexia and hyperlactatemia of tumor-bearing rats. However, INS and INS+GDP accentuated the loss of muscle mass (gastrocnemius, soleus and long digital extensor) without affecting the myostatin expression in the gastrocnemius muscle and blood corticosterone. GDP treatment did not promote beneficial effects. It can be concluded that treatment with INS (INS or INS+GDP), not with GDP, prevented fat wasting and weight loss in tumor-bearing rats without reducing tumor growth. These effects might be attributed to the reduction of lipases expression (ATGL and LHS) and increased food intake. The results show the physiological function of INS in the suppression of lipolysis induced by cachexia mediators in tumor-bearing rats.

Pioglitazone improves insulin sensitivity and reduces weight loss in Walker-256 tumor-bearing rats.

AIM: The lipogenic effect of pioglitazone (PGZ), an insulin (INS) sensitizer, is well established. However, few studies have evaluated PGZ effects in preventing weight loss in cancer. We investigated PGZ effects, alone or associated with INS, on INS resistance, cachexia and metabolic abnormalities induced by Walker-256 tumor in rats. MAIN METHODS: PGZ (5.0mg·kg-1, oral) or PGZ+INS (NPH, 1.0UI·kg-1, sc), were once-daily administered during 12days, starting on the day inoculation of Walker-256 tumor cells. Rats were separated in small (about 17g) and big (about 30g) tumor-bearing. KEY FINDINGS: Big tumor-bearing rats showed greater cachexia, blood triacylglycerol and free fatty acids and INS resistance. PGZ and PGZ+INS treatments did not change tumor growth and food intake, but reduced several abnormalities such as INS resistance, increased blood free fatty acids, retroperitoneal fat wasting and body weight loss in small tumor-bearing rats. The prevention of retroperitoneal fat wasting did not involve reduction of tumor necrosis factor-α expression increased. In big tumor-bearing rats, PGZ and PGZ+INS treatments reversed the high blood triacylglycerol and free fatty acids levels, but had no effect on other parameters. SIGNIFICANCE: PGZ and PGZ+INS improved INS peripheral sensitivity, possibly by decreasing blood free fatty acids, and reduced fat tissue wasting and body weight loss in small tumor-bearing rats. The results suggest clinical benefits of PGZ in preventing INS resistance, adipose tissue wasting and weight loss when the tumor is small, i.e., in less severe cachexia.

Celecoxib and Ibuprofen Restore the ATP Content and the Gluconeogenesis Activity in the Liver of Walker-256 Tumor-Bearing Rats.

BACKGROUND/AIMS: The main purpose of this study was to investigate the effects of celecoxib and ibuprofen, both non-steroidal anti-inflammatory drugs (NSAIDs), on the decreased gluconeogenesis observed in liver of Walker-256 tumor-bearing rats. METHODS: Celecoxib and ibuprofen (both at 25 mg/Kg) were orally administered for 12 days, beginning on the same day when the rats were inoculated with Walker-256 tumor cells. RESULTS: Celecoxib and ibuprofen treatment reversed the reduced production of glucose, pyruvate, lactate and urea from alanine as well as the reduced production of glucose from pyruvate and lactate in perfused liver from tumor-bearing rats. Besides, celecoxib and ibuprofen treatment restored the decreased ATP content, increased triacylglycerol levels and reduced mRNA expression of carnitine palmitoyl transferase 1 (CPT1), while ibuprofen treatment restored the reduced mRNA expression of peroxisome proliferator-activated receptor alpha (PPARα) in the liver of tumor-bearing rats. Both treatments tended to decrease TNFα, IL6 and IL10 in the liver of tumor-bearing rats. Finally, the treatment with celecoxib, but not with ibuprofen, reduced the growth of Walker-256 tumor. CONCLUSION: Celecoxib and ibuprofen restored the decreased gluconeogenesis in the liver of Walker-256 tumor-bearing rats. These effects did not involve changes in tumor growth and probably occurred by anti-inflammatory properties of these NSAIDs, which increased expression of genes associated with fatty acid oxidation (PPARα and CPT1) and consequently the ATP production, normalizing the energy status in the liver of tumor-bearing rats.

Early treatment with metformin induces resistance against tumor growth in adult rats.

It is known that antidiabetic drug metformin, which is used worldwide, has anti-cancer effects and can be used to prevent cancer growth. We tested the hypothesis that tumor cell growth can be inhibited by early treatment with metformin. For this purpose, adult rats chronically treated with metformin in adolescence or in adulthood were inoculated with Walker 256 carcinoma cells. Adult rats that were treated with metformin during adolescence presented inhibition of tumor growth, and animals that were treated during adult life did not demonstrate any changes in tumor growth. Although we do not have data to disclose a molecular mechanism to the preventive metformin effect, we present, for the first time, results showing that cancer growth in adult life is dependent on early life intervention, thus supporting a new therapeutic prevention for cancer.

Instant coffee extract with high chlorogenic acids content inhibits hepatic G-6-Pase in vitro, but does not reduce the glycaemia.

Coffee is the main source of chlorogenic acid in the human diet, and it contains several chlorogenic acid isomers, of which the 5-caffeoylquinic acid (5-CQA) is the predominant isomer. Because there are no available data about the action of chlorogenic acids from instant coffee on hepatic glucose-6-phosphatase (G-6-Pase) activity and blood glucose levels, these effects were investigated in rats. The changes on G-6-Pase activity and liver glucose output induced by 5-CQA were also investigated. Instant coffee extract with high chlorogenic acids content (37.8%) inhibited (p < 0.05) the G-6-Pase activity of the hepatocyte microsomal fraction in a dose-dependent way (up to 53), but IV administration of this extract did not change the glycaemia (p > 0.05). Similarly, 5-CQA (1 mM) reduced (p < 0.05) the activity of microsomal G-6-Pase by about 40%, but had no effect (p > 0.05) on glucose output arising from glycogenolysis in liver perfusion. It was concluded that instant coffee extract with high content of chlorogenic acids inhibited hepatic G-6-Pase in vitro, but failed to reduce the glycaemia probably because the coffee chlorogenic acids did not reach enough levels within the hepatocytes to inhibit the G-6-Pase and reduce the liver glucose output.

Effects of celecoxib and ibuprofen on metabolic disorders induced by Walker-256 tumor in rats.

The contribution of anti-inflammatory property of celecoxib in the improvement of metabolic disorders in cancer is unknown. The purpose of this study was to compare the effects of celecoxib and ibuprofen, non-steroidal anti-inflammatory drugs (NSAIDs), on several metabolic changes observed in Walker-256 tumor-bearing rats. The effects of these NSAIDs on the tumor growth were also assessed. Celecoxib or ibuprofen (both at 25 mg/Kg) was administered orally for 12 days, beginning on the day the rats were inoculated with Walker-256 tumor cells. Celecoxib treatment prevented the losses in body mass and mass of retroperitoneal adipose tissue, gastrocnemius, and extensor digitorum longus muscles in tumor-bearing rats. Celecoxib also prevented the rise in blood levels of triacylglycerol, urea, and lactate, the inhibition of peripheral response to insulin and hepatic glycolysis, and tended to attenuate the decrease in the food intake, but had no effect on the reduction of glycemia induced by the tumor. In addition, celecoxib treatment increased the number of Walker-256 cells with signs of apoptosis and the tumor necrosis area and prevented the tumor growth. In contrast, ibuprofen treatment had no effect on metabolic parameters affected by the Walker-256 tumor or tumor growth. It can be concluded that celecoxib, unlike ibuprofen, ameliorated several metabolic changes in rats with Walker-256 tumor due to its anti-tumor effect and not its anti-inflammatory property.

Protective effect of metformin against walker 256 tumor growth is not dependent on metabolism improvement.

BACKGROUND/AIMS: The objective of the current work was to test the effect of metformin on the tumor growth in rats with metabolic syndrome. METHODS: We obtained pre-diabetic hyperinsulinemic rats by neonatal treatment with monosodium L-glutamate (MSG), which were chronically treated every day, from weaning to 100 day old, with dose of metformin (250 mg/kg body weight). After the end of metformin treatment, the control and MSG rats, treated or untreated with metformin, were grafted with Walker 256 carcinoma cells. Tumor weight was evaluated 14 days after cancer cell inoculation. The blood insulin, glucose levels and glucose-induced insulin secretion were evaluated. RESULTS: Chronic metformin treatment improved the glycemic homeostasis in pre-diabetic MSG-rats, glucose intolerance, tissue insulin resistance, hyperinsulinemia and decreased the fat tissue accretion. Meanwhile, the metformin treatment did not interfere with the glucose insulinotropic effect on isolated pancreatic islets. Chronic treatment with metformin was able to decrease the Walker 256 tumor weight by 37% in control and MSG rats. The data demonstrated that the anticancer effect of metformin is not related to its role in correcting metabolism imbalances, such as hyperinsulinemia. However, in morphological assay to apoptosis, metformin treatment increased programmed cell death. CONCLUSION: Metformin may have a direct effect on cancer growth, and it may programs the rat organism to attenuate the growth of Walker 256 carcinoma.

Tumor necrosis factor alpha abolished the suppressive effect of insulin on hepatic glucose production and glycogenolysis stimulated by cAMP.

BACKGROUND: Tumor necrosis factor alpha (TNFα) is implicated in the development of insulin resistance in obesity, type 2 diabetes and cancer. However, its ability to modulate the action of insulin on glycogen catabolism in the liver is controversial. The aim of the present study was to investigate whether TNFα acutely affects the suppression by insulin of hepatic glucose production (HGP) and glycogenolysis stimulated by cyclic adenosine monophosphate (cAMP). METHODS: TNFα (10 µg/kg) was injected intravenously to rats and, 1 or 6h later, their livers were subjected to in situ perfusion with cAMP (3 µM), in the presence or absence of physiological (20 µU/mL) or supraphysiological (500 µU/mL) concentrations of insulin. RESULTS: The injection of TNFα, 1 or 6h before liver perfusion, had no direct effect on the action of cAMP in stimulating HGP and glycogenolysis. However, when TNFα was injected 1h, but not 6h, before liver perfusion it completely abolished (p<0.05) the suppressive effect of 20 µU/mL insulin on HGP and glycogenolysis stimulated by cAMP. Furthermore, the injection of TNFα 1h or 6h before liver perfusion did not influence the suppression of cAMP-stimulated HGP and glycogenolysis by 500 µU/mL insulin. CONCLUSION: TNFα acutely abolished the suppressive effect of physiological, but not supraphysiological, levels of insulin on HGP and glycogenolysis stimulated by cAMP, suggesting an important role of this mechanism to the increased HGP in several pathological states.

Pivotal role of cAMP in the activation of liver glycogen breakdown in high-fat diet fed mice.

AIMS: Liver glycogen catabolism was evaluated in male Swiss mice fed a high-fat diet rich in saturated fatty acids (HFD) or normal fat diet (NFD) during one week. MAIN METHODS: Liver glycogenolysis (LG) and liver glucose production (LGP) were measured either under basal or stimulated conditions (infusion of glycogenolytic agents). Thus, isolated perfused livers from HFD and NFD mice were infused with glycogenolytic agents, i.e., glucagon, epinephrine, phenylephrine, isoproterenol, adenosine-3'-5'-cyclic monophosphate (cAMP), N(6),2'-O-dibutyryl-cAMP (DB-cAMP), 8-bromoadenosine-cAMP (8-Br-cAMP) or N(6)-monobutyryl-cAMP (N6-MB-cAMP). Moreover, glycemia and liver glycogen content were measured. KEY FINDINGS: Glycemia, liver glycogen content and basal rate of LGP and LG were not influenced by the HFD. However, LGP and LG were lower (p<0.05) in HFD mice during the infusions of glucagon (1 nM), epinephrine (20 µM) or phenylephrine (20 µM). In contrast, the activation of LGP and LG during the infusion of isoproterenol (20 µM) was not different (HFD vs. NFD). Because glucagon showed the most prominent response, the effect of cAMP, its intracellular mediator, on LGP and LG was investigated. cAMP (150 µM) showed lower activation of LGP and LG in the HFD group. However, the activation of LGP and LG was not influenced by HFD whether DB-cAMP (3 µM), 8-Br-cAMP (3 µM) or N6-MB-cAMP (3 µM) were used. SIGNIFICANCE: The activation of LGP and LG depends on the intracellular availability of cAMP. It can be concluded that cAMP played a pivotal role on the activation of LG in high-fat diet fed mice.

Changes in liver gluconeogenesis during the development of Walker-256 tumour in rats.

Few studies have investigated liver gluconeogenesis in cancer and there is no agreement as to whether the activity of this pathway is increased or decreased in this disease. The aim of this study was to evaluate gluconeogenesis from alanine, pyruvate and glycerol, and related metabolic parameters in perfused liver from Walker-256 tumour-bearing rats on days 5 (WK5 group), 8 (WK8 group) and 12 (WK12 group) of tumour development. There was reduction (P < 0.05) of liver glucose production from alanine and pyruvate in WK5, WK8 and WK12 groups, which was accompanied by a decrease (P < 0.05) in oxygen consumption. Moreover, there was higher (P < 0.05) pyruvate and lactate production from alanine in the WK5 group and a marked reduction (P < 0.05) of pyruvate and urea production from alanine in the WK12 group. In addition, liver glucose production and oxygen consumption from glycerol were not reduced in WK5, WK8 and WK12 groups. Thus the, the results show inhibition of hepatic gluconeogenesis from alanine and pyruvate, but not from glycerol, on days 5, 8 and 12 of Walker-256 tumour development, which can be attributed to the metabolic step in which the substrate enters the gluconeogenic pathway.

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver.

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 µg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.