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. 2012 Sep 15;303(6):E740-51.
doi: 10.1152/ajpendo.00328.2011. Epub 2012 Jul 17.

Insulin Detemir Enhances Proglucagon Gene Expression in the Intestinal L Cells via Stimulating β-Catenin and CREB Activities

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Insulin Detemir Enhances Proglucagon Gene Expression in the Intestinal L Cells via Stimulating β-Catenin and CREB Activities

Shenghao Liu et al. Am J Physiol Endocrinol Metab. .
Free PMC article

Abstract

Insulin therapy using insulin detemir (d-INS) has demonstrated weight-sparing effects compared with other insulin formulations. Mechanisms underlying these effects, however, remain largely unknown. Here we postulate that the intestinal tissues' selective preference allows d-INS to exert enhanced action on proglucagon (Gcg) expression and the production of glucagon-like peptide (GLP)-1, an incretin hormone possessing both glycemia-lowering and weight loss effects. To test this hypothesis, we used obese type 2 diabetic db/db mice and conducted a 14-day intervention with daily injection of a therapeutic dose of d-INS or human insulin (h-INS) in these mice. The body weight of the mice after 14-day daily injection of d-INS (5 IU/kg) was decreased significantly compared with those injected with the same dose of h-INS or saline. The weight-sparing effect of d-INS was associated with significantly elevated circulating levels of total GLP-1 and reduced food intake. Histochemistry analysis demonstrated that d-INS induced rapid phosphorylation of protein kinase B (Akt) in the gut L cells of normal mice. Western blotting showed that d-INS stimulated Akt activation in a more rapid and enhanced fashion in the mouse distal ileum compared with those by h-INS. In vitro investigation in primary fetal rat intestinal cell (FRIC) cultures showed that d-INS increased Gcg mRNA expression as determined by Northern blotting and real-time RT-PCR. Consistent with these in vivo investigations, d-INS significantly increased GLP-1 secretion in FRIC cultures. Consistently, d-INS was also shown to induce rapid phosphorylation of Akt in the clonal gut cell line GLUTag. Furthermore, d-INS increased β-catenin phosphorylation, its nuclear translocation, and enhanced cAMP response element-binding protein (CREB) phosphorylation in a phosphatidylinositol 3-kinase and/or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase-sensitive manner. We suggest that the weight-sparing benefit of d-INS in mice is related to its intestinal tissues preference that leads to profound stimulation of Gcg expression and enhanced GLP-1 secretion in intestinal L cells, potentially involving the activation of insulin/β-catenin/CREB signaling pathways.

Figures

Fig. 1.
Fig. 1.
Insulin detemir (d-INS) increases serum glucagon-like peptide (GLP)-1, decreases weight gain, and reduces food intake in db/db mice. A: ip glucose tolerance test (IPGTT) performed after 14 days intervention either with PBS [control (Ctrl)], human insulin (h-INS), and d-INS injections. The areas under the glycemic curves (AUC) are shown in B. C: total GLP-1 RIA conducted in serum from db/db mice treated with d-INS or h-INS at 5 IU/kg (ip daily) or saline for 14 days. D: cumulative changes of body wt were measured during the 14-day insulin therapy. E: cumulative changes of food intake during the 14-day intervention. NS, not significant. Data are means ± SE. *P < 0.05, n = 4–5.
Fig. 2.
Fig. 2.
d-INS induces more rapid and enhanced phosphorylation (p) of protein kinase B (Akt) in distal ileum in mice. Time course of Akt phosphorylation by Western blotting using isolated distal ileum (A), brain tissue (B), muscle (C), or liver tissue (D) from CD1 mice given injections of d-INS or h-INS (1 U/kg iv) for indicated times. A′–D′: scatter plots showing densitometry analysis. E: histochemistry of isolated distal ileum from mice injected with d-INS (1 U/kg iv) for 10 min or PBS as control, dual stained for GLP-1 (red) and p-Akt (green). Arrows indicate GLP-1 positively stained cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AU, arbitrary units. Data are means ± SE. *P < 0.05 (vs. control), n = 5.
Fig. 3.
Fig. 3.
d-INS enhances proglucagon (Gcg) mRNA expression in gut L cells. A: Gcg mRNA levels in GLUTag cells treated with d-INS for indicated time points were detected by real-time RT-PCR. B: Northern blotting of GLUTag cells treated 100 nM d-INS, h-INS, or 10 μM froskolin/isobutyl methylxanthine (F/I) for 4 h. C: real-time RT-PCR analysis of Gcg mRNA levels in fetal rat intestinal cell (FRIC) cultures treated with indicated agents for 4 h. D: GLP-1 RIA conducted in FRIC cultures in the presence of indicated agents for 2 h. Data are means ± SE. *P < 0.05 and **P < 0.01, n = 3.
Fig. 4.
Fig. 4.
d-INS stimulates Akt and its downstream glycogen synthase kinase (GSK)-3 phosphorylation in GLUTag cells. Cells were starved overnight and treated with the indicated insulin at indicated dosages for the indicated time. Time courses of 100 nM d-INS (A) and h-INS (B) on Akt phosphorylation were examined by Western blotting. Dose courses of d-INS (C) and h-INS (D) on Akt phosphorylation. Time course of d-INS (E) and h-INS (F) on GSK-3 phosphorylation was conducted by Western blotting. The black lines in the immunoblots in E and F signify that blots have been spliced, from the same membrane, to indicate the most representative Western blots corresponding to the bar graphs. Data are means ± SE. *P < 0.05, n = 3–4.
Fig. 5.
Fig. 5.
d-INS stimulates β-catenin (β-cat) Ser675 phosphorylation and nuclear translocation. Western blotting of time course of 100 nM d-INS (A) and h-INS (B) on β-cat phosphorylation. Dose courses of d-INS (C) and h-INS (D) on β-cat phosphorylation (10 min). Time course of β-cat nuclear detection in GLUTag cells (E and F). Bar graphs show the densitometry analysis. Data are means ± SE. *P < 0.05, n = 4–5.
Fig. 6.
Fig. 6.
d-INS stimulates cytosolic and nuclear extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in GLUTag cells. Western blotting in cell lysates of GLUTag treated with 100 nM d-INS for indicated times (A), or treated with d-INS at indicated dose for 10 min (B), using relevant antibodies as indicated. Time course of d-INS on cytosolic and nuclear ERK phosphorylation (C and D). Bar graphs show the densitometry analysis. RU, relative units; t-ERK, total ERK. Data are means ± SE. *P < 0.05, n = 3.
Fig. 7.
Fig. 7.
d-INS stimulates cAMP response element-binding protein (CREB) phosphorylation, which is phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK)/ERK dependent in GLUTag cells. Cells were serum starved overnight and treated with d-INS at indicated concentrations for 10 min (A) or 100 nM d-INS for indicated times (B). Cell lysates were subjected to Western blotting. C: serum-starved GLUTag cells were treated with 100 nM d-INS for 10 min in the presence of an indicated inhibitor (PD, 50 μM; LY, 50 μM), and nuclear fractions were subjected to Western blotting using Histone H3 as loading controls. Bar graphs show the densitometry analysis. Data are means ± SE. *P < 0.05 and #P < 0.05 (vs. control), n = 3–4.
Fig. 8.
Fig. 8.
Hypothetic model showing the roles of PI3-K and MAPK/ERK pathways in transducing the stimulatory insulin signal to Gcg expression. Activation of the insulin receptor stimulates the phosphorylation of β-cat at Ser675 and its nuclear translocation via PI3-K- and MAPK/ERK-dependent pathways. Insulin also stimulates nuclear CREB phosphorylation via an MAPK/ERK-dependent fashion. The downstream effectors of β-cat and CREB that initiate gene transcription are not shown. Solid lines, established pathways; dotted lines, putative pathways.

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