Supplementary Information Akt regulates hepatic metabolism by suppressing a Foxo1 dependent global inhibition of adaptation to nutrient intake Mingjian Lu 1, Min Wan 1, Karla F. Leavens 1, Qingwei Chu 1, Bobby R. Monks 1, Sully Fernandez 1, Rexford S. Ahima 1, Kohjiro Ueki 2, C. Ronald Kahn 3, and Morris J. Birnbaum 1, * 1 The Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 1914, USA 2 Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 3 Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 2215, USA * Address correspondence to: Morris J. Birnbaum, M.D. Ph.D. Room 12-123 TRC 34 Civic Center Blvd., Bldng. 421 Philadelphia PA 1914 email: birnbaum@mail.med.upenn.edu
Supplementary Figure 1 a b 2LWT 2LKO 2LWT 2LKO 2LWT 2LKO p Akt S473 Akt2 Tubulin Fasted Refed Insulin Relative p Akt S473 Fasted 2LWT 2LKO Refed Insulin Supplementary Figure 1. Akt phosphorylation in the control and 2LKO livers four hours after feeding and 2 minutes after insulin injection. 2LWT or 2LKO mice were fasted overnight (fasted), followed by four hours feeding (refed) or injection with insulin at 2 mu g 1 (insulin). For insulin-injected mice, livers were harvested 2 minutes after injection. Liver lysates were subjected to Western blot with indicated antibodies (a). The p Akt S473 image intensity was quantitated with NIH Image J program, and shown in (b). Assuming that all Akt1 (16% of total Akt) was phosphorylated after insulin injection, we estimated that less than 4% of total Akt was phosphorylated 4 hours after feeding in the control livers.
Supplementary Figure 2 Glycogen (mg g 1 liver) 8 6 4 2 2LWT Fasted Refed 2LKO * DLWT DLKO *** Supplementary Figure 2. Defective postprandial glycogen storage in the DLKO livers. Mice were fasted overnight, or fasted overnight and refed for four hours. Glycogen content in the livers was measured. The glycogen in fed livers was slightly but significantly lower in the 2LKO livers than the 2LWT livers. (* P <.5, *** P <.1).
Supplementary Figure 3 Glucose uptake, muscle (nmol g 1 min 1 ) 25 2 15 1 5 DLWT *** DLKO glucose uptake, WAT (nmol g 1 min 1 ) 2 15 1 5 DLWT *** DLKO Supplementary Figure 3. Defective glucose uptake in skeletal muscle and adipose tissues of the DLKO mice. After the hyperinsulinemic euglycemic clamp, 2 deoxyglucose uptake in skeletal muscle and epididymal adipose tissues (WAT) were measured. (*** P <.1).
Supplementary Figure 4 a Relative expression Foxo1 b 2 1 Control Foxo1 / c Fasting blood glucose (mg dl 1 ) d 1 8 6 4 2 Control *** Foxo1 / Control Foxo1 / Foxo1 Akt2 Feeding blood glucose (mg dl 1 ) 2 15 1 5 Control Foxo1 / Supplementary Figure 4. Acute deletion of Foxo1 in mouse livers resulted in fasting hypoglycemia. Foxo1 loxp/loxp mice 1 were injected with AAV-Tbg-Cre (Foxo1 / ) or AAV-Tbg-GFP (control). (a) Two weeks after virus injection, mice were sacrificed, total RNA was isolated from whole livers and Foxo1 mrna was quantitated with real time PCR as described 2 (n = 8). In (b), protein extracts from hepatocytes isolated from the control mice and Foxo1 / mice were resolved in a SDS PAGE gel and probed for Foxo1 and Akt2. (c) Blood glucose level after overnight fasting (n = 16, 17, *** P <.1). (d) Blood glucose level after overnight fasting followed by four hours feeding (n = 7, 9).
Supplementary Figure 5 Glycogen (mg g 1 liver) *** 4 Fasted Refed 3 NS 2 1 LWT DLKO TLKO Supplementary Figure 5. Defective postprandial glycogen storage in the DLKO and the TLKO livers. Mice were fasted overnight, or fasted overnight and refed for four hours. Liver glycogen content was measured. The difference in glycogen content after feeding between the DLKO livers and the TLKO livers did not reach statistical significance (*** P <.1, NS: not significant, n = 7,8).
Supplementary Figure 6 G d 6,6 / G d 2 8 6 4 2 LWT DLKO TLKO 5 1 15 Time (min) Supplementary Figure 6. Defective glucose cycling in the DLKO livers but not in the TLKO livers. The glucose G 6 P cycling was measured as previously reported 3. Briefly, mice were fasted overnight, orally given a mixture of glucose either singlelabeled at 2 position or double-labeled at 6-position with deuterium. If glucose goes through the glucose à glucokinaseà G 6 P à G 6 phosphatase à glucose cycle, the label at 2 position is preferentially lost, increasing the ratio of d 6,6 double label glucose to the d 2 single-labeled glucose. In the experiment, serum was collected at 3, 6, and 12 minutes after glucose gavage. The enrichment of the d 6,6 double-label glucose and the d 2 single-labeled glucose was determined by Mass Spectrometry. As shown in the figure, the glucose G 6 P cycling was indistinguishable between the LWT livers and the TLKO livers and undetected in the DLKO mice.
Supplementary Figure 7 WT Akt3 / Brain lysate Chow + + + LWT DLKO TLKO Liver lysate Akt3 Akt2 Foxo1 Tubulin Supplementary Figure 7. Akt3 was not increased in either the DLKO livers or the TLKO livers. Liver lysates from the control (LWT), DLKO, or the TLKO mice were subjected to SDS PAGE and Western blotting for Akt3, Akt2, Foxo1 and tubulin (as loading control). Brain lysates with the same amount of total protein from the wild type mice (WT) or Akt3 knockout mice (Akt3 / ) were used as positive control and negative control, respectively, for Akt3.
Supplementary Figure 8 Relative expression Sgk1 8 6 4 2 LWT DLKO TLKO Fasted Refed Supplementary Figure 8. Sgk1 expression increased in the DLKO but not the TLKO livers. Mice in each genotype were fasted overnight. For the refed groups, mice were fed for four hours after fasting. Relative expression of Sgk1 in livers was quantitated by real time PCR.
Supplementary Figure 9
Supplementary Figure 9. Unrestrained Foxo1 activity suppresses the metabolic transition from fasting to feeding. Heatmap of the expression of the 298 metabolically responsive genes in control, DLKO and TLKO livers in response to the fasting to feeding transition. Column 1: LWT, fasted. Column 2: LWT, fed. Column 3: DLKO, fasted. Column 4: DLKO, fasted. Column 5: TLKO, fasted. Column 6: TLKO, fed. Note that in the DLKO livers, the expression profile in both the fasted and fed states resembled that in the fasted state in the control livers, and the block of the transition from fasting to feeding is at least partially reversed in the TLKO livers. The metabolically responsive genes were defined as those with at least 2 fold change from fasting to feeding.
Supplementary Figure 1 Fasted Fed Irs1, Irs2 DKO, log 2 (DKO/WT) Akt1, Akt2 DLKO, log 2 (DLKO/LWT) Supplementary Figure 1. Correlation in gene expression between the Akt1/Akt2 DLKO livers and the Irs1/Irs2 DKO livers under fasting and fed conditions. The ratio of gene expression in the Akt1;Akt2 DLKO to control livers was compared the ratio of Irs1;Irs2 DKO to control livers under fasting and fasting/refed conditions (AffyexpressE- MEXP 1649). The X axis and the Y axis are the M values ( log 2 (test/control) ) from the arrays for Akt DLKO and Irs DKO, respectively. The graph is a density plot which shows the density of points when they are close together, the dark dots indicate that these is only one single point in a density drop and were used to highlight individual points when they are farther apart. The R values in the graph are the Pearson's correlation value. The large number of genes in the upper right and lower left quadrants indicates that the deletion of Akt and Irs in liver altered expression of the same genes and the proximity to the diagonal shows that the magnitudes of the changes were similar. The correlation is statistically significant (P < 2e 16).
Supplementary Figure 11 Supplementary Figure 11. Schematic illustration of regulation of liver glucose metabolism by Akt and Foxo1. In the canonical model, Akt activation-dependent Foxo1 suppression is responsible for the decreased expression of Pck1 and G6pc and liver glucose output. In the proposed new model, in addition to the Aktà Foxo1 axis, there is another Akt and Foxo1 independent pathway that regulates liver glucose output and the expression of G6pc and Pck1 in response to insulin and nutrients, and this pathway is repressed by unrestrained Foxo1 activation. In the absence of Akt, Foxo1 increases liver glucose output by directly inducing G6pc and Pck1 expression and by repressing the parallel pathway. In livers lacking both Akt and Foxo1, the parallel pathway is fully functional and sufficient to signal the response to feeding, both in terms of the expression of G6pc and Pck1 and glucose output. Ex vivo, primary hepatocytes behave as would be predicted if only the canonical pathway exists.
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