Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity.
ABSTRACT: 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 ?1 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 ?1 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.
Project description: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.
Project description:The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.
Project description: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 ?1 subunit, but whether salsalate requires AMPK-?1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-?1-knockout (AMPK-?1KO) 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-?1KO 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.
Project description:The deregulation of lipid metabolism is a hallmark of tumor cells, and elevated lipogenesis has been reported in prostate cancer. Metformin, a drug commonly prescribed for type II diabetes, displays antitumor properties. Here, we show that metformin inhibits lipogenesis in several prostate cancer cell lines. In LNCaP cells, this effect parallels the decrease of key lipogenic proteins: ACC (acetyl-CoA carboxylase), FASN (fatty acid synthase) and SREBP1c (sterol regulatory element binding protein-1c), whereas there is no modification in DU145 and PC3 cells. Despite the relatively high level of lipogenic proteins induced by the overexpression of a constitutively active form of SREBP1c or treatment with androgens, metformin is still able to inhibit lipogenesis. Metformin does not alter the concentration of malonyl-CoA (the fatty acid precursor), and it only slightly decreases the NADPH levels, which is a co-factor required for lipogenesis, in LNCaP. Finally, we show that the inhibitory effect of metformin on lipogenesis is primarily due to a cellular energy deficit. Metformin decreases ATP in a dose-dependent manner, and this diminution is significantly correlated with the inhibition of lipogenesis in LNCaP and DU145. Indeed, the effect of metformin is linked to changes in the ATP content rather than the regulation of protein expression. Our results describe a new mechanism of action for metformin on prostate cancer metabolism.
Project description:The metabolic pathway of de novo lipogenesis is frequently upregulated in human liver tumours, and its upregulation is associated with poor prognosis. Blocking lipogenesis in cultured liver cancer cells is sufficient to decrease cell viability; however, it is not known whether blocking lipogenesis in vivo can prevent liver tumorigenesis. Herein, we inhibit hepatic lipogenesis in mice by liver-specific knockout of acetyl-CoA carboxylase (ACC) genes and treat the mice with the hepatocellular carcinogen diethylnitrosamine (DEN). Unexpectedly, mice lacking hepatic lipogenesis have a twofold increase in tumour incidence and multiplicity compared to controls. Metabolomics analysis of ACC-deficient liver identifies a marked increase in antioxidants including NADPH and reduced glutathione. Importantly, supplementing primary wild-type hepatocytes with glutathione precursors improves cell survival following DEN treatment to a level indistinguishable from ACC-deficient primary hepatocytes. This study shows that lipogenesis is dispensable for liver tumorigenesis in mice treated with DEN, and identifies an important role for ACC enzymes in redox regulation and cell survival.
Project description:Inhibiting lipogenesis prevents hepatic steatosis in rodents with insulin resistance. To determine if reducing lipogenesis functions similarly in humans, we developed MK-4074, a liver-specific inhibitor of acetyl-CoA carboxylase (ACC1) and (ACC2), enzymes that produce malonyl-CoA for fatty acid synthesis. MK-4074 administered to subjects with hepatic steatosis for 1 month lowered lipogenesis, increased ketones, and reduced liver triglycerides by 36%. Unexpectedly, MK-4074 increased plasma triglycerides by 200%. To further investigate, mice that lack ACC1 and ACC2 in hepatocytes (ACC dLKO) were generated. Deletion of ACCs decreased polyunsaturated fatty acid (PUFA) concentrations in liver due to reduced malonyl-CoA, which is required for elongation of essential fatty acids. PUFA deficiency induced SREBP-1c, which increased GPAT1 expression and VLDL secretion. PUFA supplementation or siRNA-mediated knockdown of GPAT1 normalized plasma triglycerides. Thus, inhibiting lipogenesis in humans reduced hepatic steatosis, but inhibiting ACC resulted in hypertriglyceridemia due to activation of SREBP-1c and increased VLDL secretion.
Project description:Fibroblast growth factor 21 (FGF21) shows great potential for the treatment of obesity and type 2 diabetes, as its long-acting analogue reduces body weight and improves lipid profiles of participants in clinical studies; however, the intracellular mechanisms mediating these effects are poorly understood. AMP-activated protein kinase (AMPK) is an important energy sensor of the cell and a molecular target for anti-diabetic medications. This work examined the role of AMPK in mediating the glucose and lipid-lowering effects of FGF21.Inducible adipocyte AMPK ?1?2 knockout mice (i?1?2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received long-acting FGF21 for two weeks.Consistent with previous studies, FGF21 treatment significantly reduced body weight, adiposity, and liver lipids in HFD fed mice. To add, FGF21 improved circulating lipids, glycemic control, and insulin sensitivity. These effects were independent of adipocyte AMPK and were not associated with changes in browning of white (WAT) and brown adipose tissue (BAT). Lastly, we assessed whether FGF21 exerted its effects through the AMPK/ACC axis, which is critical in the therapeutic benefits of the anti-diabetic medication metformin. ACC DKI mice had improved glucose and insulin tolerance and a reduction in body weight, body fat and hepatic steatosis similar to WT mice in response to FGF21 administration.These data illustrate that the metabolic improvements upon FGF21 administration are independent of adipocyte AMPK, and do not require the inhibitory action of AMPK on ACC. This is in contrast to the anti-diabetic medication metformin and suggests that the treatment of obesity and diabetes with the combination of FGF21 and AMPK activators merits consideration.
Project description:Estradiol has been used to prevent metabolic diseases, bone loss and menopausal symptoms, even though it might raise the risk of cancer. Metformin is usually prescribed for type 2 diabetes mellitus and lowers food intake and body mass while improving insulin resistance and the lipid profile. Ovariectomized rats show increased body mass, insulin resistance and changes in the lipid profile. Thus, the aim of this work was to evaluate whether metformin could prevent the early metabolic dysfunction that occurs early after ovariectomy. Female Wistar rats were divided into the following groups: SHAM-operated (SHAM), ovariectomized (OVX), ovariectomized + estradiol (OVX + E2) and ovariectomized + metformin (OVX + M). Treatment with metformin diminished approximately 50% of the mass gain observed in ovariectomized animals and reduced both the serum and hepatic triglyceride levels. The hepatic levels of phosphorylated AMP-activated protein kinase (pAMPK) decreased after OVX, and the expression of the inactive form of hepatic acetyl-CoA carboxylase (ACC) was also reduced. Metformin was able to increase the levels of pAMPK in the liver of OVX animals, sustaining the balance between the inactive and total forms of ACC. Estradiol effects were similar to those of metformin but with different proportions. Our results suggest that metformin ameliorates the early alterations of metabolic parameters and rescues hepatic AMPK phosphorylation and ACC inactivation observed in ovariectomized rats.
Project description:Cetuximab inhibits HIF-1-regulated glycolysis in cancer cells, thereby reversing the Warburg effect and leading to inhibition of cancer cell metabolism. AMP-activated protein kinase (AMPK) is activated after cetuximab treatment, and a sustained AMPK activity is a mechanism contributing to cetuximab resistance. Here, we investigated how acetyl-CoA carboxylase (ACC), a downstream target of AMPK, rewires cancer metabolism in response to cetuximab treatment. We found that introduction of experimental ACC mutants lacking the AMPK phosphorylation sites (ACC1_S79A and ACC2_S212A) into head and neck squamous cell carcinoma (HNSCC) cells protected HNSCC cells from cetuximab-induced growth inhibition. HNSCC cells with acquired cetuximab resistance contained not only high levels of T172-phosphorylated AMPK and S79-phosphorylated ACC1 but also an increased level of total ACC. These findings were corroborated in tumor specimens of HNSCC patients treated with cetuximab. Cetuximab plus TOFA (an allosteric inhibitor of ACC) achieved remarkable growth inhibition of cetuximab-resistant HNSCC xenografts. Our data suggest a novel paradigm in which cetuximab-mediated activation of AMPK and subsequent phosphorylation and inhibition of ACC is followed by a compensatory increase in total ACC, which rewires cancer metabolism from glycolysis-dependent to lipogenesis-dependent.
Project description:Interleukin-6 (IL-6) has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD) in wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice. Additionally, HFD-fed IL-6(-/-) mice were also chronically treated with recombinant IL-6 (rIL-6). Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1) and signal transducer and activator of transcription-3 (STAT3), increased AMP kinase phosphorylation (p-AMPK), and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). The HFD-fed IL-6(-/-) mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACC?/?), FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6 -/-: mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACC?/?, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis.