Metformin Inhibits Hepatic mTORC1 Signaling via Dose-Dependent Mechanisms Involving AMPK and the TSC Complex

Abstract

Metformin is the most widely prescribed drug for the treatment of type 2 diabetes. However, knowledge of the full effects of metformin on biochemical pathways and processes in its primary target tissue, the liver, is limited. One established effect of metformin is to decrease cellular energy levels. The AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) are key regulators of metabolism that are respectively activated and inhibited in acute response to cellular energy depletion. Here we show that metformin robustly inhibits mTORC1 in mouse liver tissue and primary hepatocytes. Using mouse genetics, we find that at the lowest concentrations of metformin that inhibit hepatic mTORC1 signaling, this inhibition is dependent on AMPK and the tuberous sclerosis complex (TSC) protein complex (TSC complex). Finally, we show that metformin profoundly inhibits hepatocyte protein synthesis in a manner that is largely dependent on its ability to suppress mTORC1 signaling.

Keywords: AMPK; TSC1; TSC2; hepatocytes; liver; mTOR; mTORC1; metformin; protein synthesis; tuberous sclerosis complex.

Figure 1. Metformin suppresses hepatic mTORC1 signaling in an AMPK-dependent manner

(A) Male mice (age 10 weeks) were fasted overnight then left unfed (Fast; n=2) or refed for 6 h, with saline, 200 or 250 mg/kg metformin treatment (n=3 per treatment) for the last 1 h. See Figure S1 for measurements of plasma metformin concentrations. (B) Female AMPKα1^fl/fl/α2^fl/fl or L-AMPKα1/α2-DKO mice (age 6 mos) were fasted overnight, refed for 2 h and treated with saline or 250 mg/kg metformin for the last 1 h (n=4 per treatment). See Figure S1 for supporting data with a male cohort age 12 weeks. (C,D) Primary hepatocytes were treated (C) for 5 h with indicated doses of metformin or 20 nM rapamycin (Rap) or (D) with 0.5 mM metformin for the indicated times. (E) Primary hepatocytes from AMPKα1^fl/fl/α2^fl/fl or L-AMPKα1/α2-DKO mice were treated with the indicated concentrations of metformin for 2 or 5 h.

Figure 2. Mice with liver-specific disruption of the TSC complex display metformin-resistant mTORC1 signaling

(A) Male TSC1^fl/fl or LTsc1KO mice (age 10 weeks) were fasted overnight then refed for 6 h and treated with saline or metformin for the last 1 h (n=2 per condition). (B) Male AMPKα1^fl/fl/α2^fl/fl or L-AMPKα1/α2-DKO (age 5 mos) mice were treated as in (A). (C) Female TSC1^fl/fl or LTsc1KO mice (age 12 week) were treated as in (A) with saline, 50, 100, or 200 mg/kg metformin (n=3 per condition). (D) Female mice (age 14 weeks) treated as in (A) with saline or 250 mg/kg metformin (n=3 per condition).

Figure 3. Low dose metformin suppresses hepatocyte mTORC1 signaling in a TSC complex-dependent manner

(A–D) Primary hepatocytes from TSC1^fl/fl or LTsc1KO mice were serum-starved overnight and treated (A–C) for 5 h with increasing doses of metformin together with (A) no stimulation, (B) 100 nM insulin, or (C) 5% FBS, or (D) with a time course of treatment with 1 mM metformin following a 15-min pretreatment with 100 nM insulin. Quantitation of P-S6 band intensities via densitometry is shown in graphical format below each panel. (E) Primary hepatocytes from TSC1^fl/fl mice were infected with adenovirus expressing GFP or Cre 36 h prior to treatment with 2 mM metformin for 5 h. (F) Oxygen consumption rates (OCR) of primary hepatocytes from TSC1^fl/fl or LTsc1KO mice were measured for 1 h in the presence of 1 mM metformin and are graphed as the mean ± SEM OCR as a percent of untreated samples measured in parallel (n=4 experiments).

Figure 4. Metformin inhibits protein synthesis in hepatocytes through the TSC complex

(A) Primary hepatocytes from TSC1^fl/fl mice were treated with 1 mM metformin for the indicated times, and mRNA 5’-cap-binding complexes were isolated from cell lysates with m⁷GTP-agarose and analyzed by immunoblotting. (B) Primary hepatocytes from TSC1^fl/fl and LTsc1KO mice were treated with 1 mM metformin for 5 h and analyzed as in (A). (C and D) Effects of metformin on hepatocyte protein synthesis. Primary hepatocytes from TSC1^fl/fl and LTsc1KO mice were pretreated with insulin for 20 min followed by treatment in the presence or absence of 1 mM metformin for 5 h, with a pulse label of [³⁵S]-methionine for the final 20 min. A representative autoradiogram from 5 independent experiments is shown. (D) Individual lanes from autoradiograms were quantified by densitometry and are graphed as mean ± SEM % incorporation relative to untreated cells (n=5 independent experiments). *P < 0.05 by Student’s two-tailed t-test. (E) Primary human hepatocytes were treated with the indicated doses of metformin for 5 h and, for assaying protein synthesis, were radiolabeled as in (C). Signaling was assessed by immunoblots, protein synthesis by autoradiogram, and total protein for this assay by Ponceau S staining. (F) Model of dose-dependent, differential regulation of mTORC1 in response to increasing metformin concentrations.