Insulin promotes proliferation and fibrosing responses in activated pancreatic stellate cells

Abstract

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high-fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia, and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared with normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 mo similarly displayed increasing peri-islet fibrosis compared with mice fed control diet. Both quiescent and activated PaSC coexpress insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling, and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data support the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.

Keywords: diabetes; insulin; obesity; pancreatic fibrosis; pancreatic stellate cells.

Copyright © 2016 the American Physiological Society.

Figures

Fig. 1.

Type-2 diabetes patients display extensive collagen deposition and stellate cell activation in pancreatic areas surrounding islets. Paraffin-embedded pancreatic tissue specimens were obtained from cadaveric tissues from organ donors at the City of Hope Medical Institute (Duarte, CA). Serial sections were analyzed for collagen deposition by Masson's trichrome staining (A, B, D, and E), and for insulin and α-SMA by immunofluorescence (C and F). The clinical and demographic data of the organ donors is presented in Table 1. A and D show representative photomicrographs of Masson's trichrome-stained pancreas tissues from organ donors without pancreas pathology (normal; A) and organ donors with type-2 diabetes (T2DM; D). Collagens are visible as the blue stain; scale bar = 100 μm. Dotted boxes in A and D indicate areas with two examples of collagen deposition surrounding islets illustrated at higher magnification in B and E, respectively; Scale bar = 100 μm. C and F: serial pancreatic tissue sections were stained with antibodies against insulin (beta cells in islets; red) and α-SMA (activated stellate cells; green); nuclei were stained with Dapi (blue). C and F illustrate boxed regions in B and E, respectively; scale bar = 20 μm. G: bar graph shows the percentage of collagen-stained area (Masson's trichrome staining) in pancreas sections from normal and T2DM organ donors. Seven randomly selected high-power fields were quantified and averaged to obtain the value for each specimen; n = 3 organ donors for each group. Data in graph are means ± SE; *P < 0.05 compared with normal.

Fig. 2.

High-fat diets promote collagen deposition in areas surrounding islets in mouse pancreas. A and B: light-field photomicrographs of Sirius red-stained pancreatic tissue sections of mice fed control chow (CD) or high-fat, high-calorie diet (HFCD) for 12 mo. Collagens are visible as bright red stain. Arrows in B indicate collagen deposition in peri-islet and peri-acinar areas; “is”, islet; “v” blood vessel. The boxed regions are shown at a higher magnification in C and D. Scale bar = 100 μm. C and D: serial pancreatic tissue sections were stained with antibodies against insulin (beta cells in islets; red staining) and α-SMA (a marker of activated stellate cells; green staining); nuclei were stained with Dapi (blue staining). C and D illustrate boxed regions in A and B, respectively; scale bar = 25 μm. E: bar graph shows the percentage of collagen-stained area (Sirius red staining) relative to the total field in pancreas sections from mice fed CD or HFCD for 3, 6, 9, and 12 mo. Seven to ten randomly selected high-power fields were quantified and averaged to obtain the value for each animal; n = 3–4 for each experimental group. Data in graph are means ± SE; *P < 0.05 compared with CD fed mice.

Fig. 3.

Quiescent and activated mouse PaSC express insulin (IR) and IGF (IGF-1R) receptors. A: freshly isolated, quiescent mouse PaSC (mPaSC) were cultured for 48 h (day 3) or 96 h (day 5) in DMEM/F12 medium (17.5 mM glucose) containing 10% FBS without or with 100 nM insulin. IR and IGF-1R mRNA expression were determined by qPCR. B: quiescent and culture-activated mPaSC (untreated or insulin-treated) preferentially expressed the IR-A isoform compared with the IR-B isoform. Quiescent mPaSC were treated as indicated in A. mRNA expression of the IR isoforms IR-A and IR-B was measured by qPCR. C and D: insulin modulates IR and IGF-1R expression in activated mPaSC. Activated mPaSC were cultured in DMEM/F12 medium with 10% FBS for 48 h. After 4 h serum starvation, cells were treated with insulin (0–100 nM) for up to 48 h in 1% FBS DMEM/F12 medium. Expression levels of IR and IGF-1R were analyzed by qPCR. Data in graphs are means ± SE; data are representative of 3 independent experiments; *P < 0.05 vs. 0 nM insulin.

Fig. 4.

Insulin does not induce activation of quiescent, freshly isolated mouse PaSC but promotes fibrosing responses. Freshly isolated mPaSC were cultured for 5 days in 1% FBS DMEM/F12 media containing 5 mM or 25 mM glucose either alone or with 100 nM insulin. Cells cultured in 10% FBS DMEM/F12 medium (17.5 mM glucose) were used as positive control for cell activation. At day 5, cells were formaldehyde-fixed and stained for α-SMA (a marker of cell activation) and Hoechst 33342 to visualize nuclei. Nuclei (A) and α-SMA positive cells (B) were counted in at least 10 randomly selected fields under low-power magnification (10X). The percentage of α-SMA positive cells was determined by the ratio of α-SMA and Hoechst-positive cells. Results are presented as means ± SE; 2 independent experiments. C: freshly isolated mPaSC were cultured for 48 h (day 3) or 96 h (day 5; fully activated) in 10% serum media with/without 100 nM insulin. Expression levels of collagen (alpha-1 type I; COL1A1) and fibronectin (FN1) mRNA were measured by qPCR. 18S rRNA was used as an internal control. Data in graph represent means ± SE from 3 independent experiments; *P < 0.05 vs. 0 nM insulin.

Fig. 5.

Insulin induces rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, and activation of Akt/mTOR/p70S6K signaling in activated PaSC. imPaSCs were cultured for two days in DMEM containing 5 mM glucose and 10% FBS, starved in serum-free medium for 4 h, and then treated with various concentrations (0–100 nM) of insulin for 30 min (A) or with 100 nM insulin for the times shown (0–60 min) (B). Levels of phosphorylated or total IR, IGF-1R/IR, Akt/mTOR/p70S6K, ERK1/2, and SAPK/JNK were measured in cell lysates by Western blotting. β-Actin was used as loading control. Similar results were obtained using primary mPaSC (not shown). C and D: graphs show protein levels of phosphorylated IGF-1R/IR determined by densitometry. Bars represent means ± SE from 3 independent experiments; *P < 0.05 compared with basal control.

Fig. 6.

Insulin-induced activation of Akt/mTOR/p70S6K and ERK signaling pathways are more pronounced in PaSC cultured in 25 mM glucose compared with 5 mM glucose. imPaSCs were cultured in DMEM media containing 5 mM glucose and 10% FBS for 2 days, starved in serum-free medium for 4 h, and then cultured in 5 mM glucose- or 25 mM glucose-containing media with or without 100 nM insulin for various periods of time (1–24 h). A: cell lysates were analyzed for the indicated targets, and representative immunoblots are shown in A. β-Actin was used as loading control. B: graphs show protein levels of phosphorylated IGF-1R/IR, p70S6K, Akt, and ERK determined by densitometry. As indicated in the graphs, insulin-induced Akt and ERK phosphorylation was more pronounced in cells cultured in media containing 25 mM glucose compared with media containing 5 mM glucose. Data in graphs represent means ± SE from 3 independent experiments.

Fig. 7.

Insulin stimulates fibrosing responses and cell proliferation in mouse immortalized PaSCs. imPaSC were cultured in 10% serum DMEM media containing 5 mM glucose, starved in serum-free medium for 4 h, and then stimulated for the indicated times with 100 nM insulin in 5 mM or 25 mM glucose-containing media (medium was changed every day with new additions of insulin). A: after 24 h incubation, expression levels of fibronectin (FN1) and α-SMA (ACTA2) were assessed by qPCR. 18S rRNA was used as an internal control. Data in graph represent means ± SE from 3 independent experiments; *P < 0.05 vs. 5 mM glucose without insulin; #P < 0.05 vs. 25 mM glucose without insulin. B: cell proliferation after 72 h stimulation with 100 nM insulin and/or 25 mM glucose was determined by MTT assay. Data in graphs represent means ± SE; n = 3 independent experiments. *P < 0.05 vs. 5 mM glucose without insulin; #P < 0.05 vs. 25 mM glucose without insulin.

Fig. 8.

KU63794 (KU) inhibits insulin-induced Akt/mTOR signaling in PaSCs. imPaSC were cultured in 10% serum DMEM containing 5 mM glucose for 2 days, starved in serum-free medium for 4 h, and then treated for the indicated times in 1% FBS DMEM with or without 100 nM insulin and in the presence or absence of the mTOR inhibitor KU at 1 μM. A: cell lysates were analyzed for the indicated targets, and representative immunoblots are shown. α-SMA was used as loading control. B: the graph shows levels of phosphorylated p706SK Thr389 and Akt Ser473 determined by densitometry. As indicated in the graphs, insulin-induced Akt phosphorylation and, especially, p70S6K phosphorylation were effectively reduced by preincubation with KU. Data in the graphs are representative of 2 independent experiments.

Fig. 9.

mTOR inhibition blocks insulin-induced fibrosing responses and cell proliferation in activated PaSC. imPaSC were cultured in 10% serum DMEM containing 5 mM glucose for 2 days, serum starved, preincubated for 30 min with or without KU and then stimulated with or without insulin for the times shown. A: cellular protein lysates were analyzed by Western blot for fibrotic proteins fibronectin (FN) or collagen or the catalytic subunit of prolyl hydroxylase (P4HA2), an important enzyme in collagen biosynthesis. α-SMA was tested as a marker of activation. β-Actin was also analyzed as a loading control. B: multiple experiments of the type shown in A at the 48-h time point were quantitated by densitometry. C: cell proliferation in cells grown as in A was measured by MTT assay. Data in graphs (B and C) represent the means ± SE; n = 3. *P < 0.05 relative to no-insulin control, 48 h. #P < 0.05 relative to no-insulin control, 72 h.

Fig. 10.

The mTOR inhibitor KU63794 (KU) dose-dependently inhibits Akt/mTOR signaling and proliferation in activated PaSC. Culture-activated mPaSCs were cultured for 2 days in 10% FBS DMEM, starved in serum-free medium for 4 h, and then treated in 5% FBS DMEM for 24 or 48 h with KU at the indicated concentrations. KU at 1 and 5 μM effectively blocked Akt/mTOR signaling but not ERK activation (A) and attenuated the cell proliferation response (B) in mPaSC. Data in the graph represent means ± SE from n = 3 independent experiments. *P < 0.05 vs. 0 μM KU.