Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB

Abstract

Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of type 2 diabetes (T2D). Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of antiapoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and pancreatic and duodenal homeobox-1 (PDX-1) expression. Chronic hyperlipidemia significantly diminished cyclic adenosine monophosphate (cAMP) production, protein kinase A (PKA) activation, cAMP-responsive element binding protein (CREB) phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol-stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade.

Fig. 1

Kaempferol protects β-cells against palmitate-induced apoptosis. INS-1E β-cells were incubated in RPMI1640 medium containing 0.5 mM palmitate (P) or vehicle with or without various concentrations of kaempferol (K; 0.1, 1, 10 μM) for 4 d. Cell viability was assessed by an MTS based assay (A) and by measuring intracellular ATP production (B). Apoptotic cells as determined by DNA fragmentation were quantified using a HT TiterTACS™ assay kit (C), and enzymatic activity of caspase-3 in cell extracts was measured (D). Values are mean ± SEM obtained from four independent experiments performed in triplicate. *, p<0.05 vs. normal control; #, p<0.05, ##, p<0.01 vs. Palmitate-alone treated cells.

Fig. 2

Kaempferol protects human islets against palmitate-induced apoptosis. Human islets (200 islets/well) were incubated in RPMI1640 medium containing 0.5 mM palmitate (P) with or without various concentrations of kaempferol (K; 0.01, 0.1, 1, 10 μM) for 4 d. Islet viability was assessed by an MTS based assay (A) and by measuring intracellular ATP production (B). Apoptotic cells were quantified with a TUNEL assay kit (C), and enzymatic activity of caspase-3 in cell extracts was measured (D). Values are mean ± SEM. obtained from three independent experiments performed in triplicate. *, P<0.05 vs. normal control; *, p<0.05, p<0.01 vs. palmitate-alone treated islets.

Fig. 3

Kaempferol restores palmitate-mediated reductions in Akt and Bcl-2 protein expression. INS-1E cells (A and B) and islets (C and D) were treated as described in Fig. 1. The levels of Akt and Bcl-2 protein in cell protein extracts was measured by immunoblot and normalized to β-actin content. Data are mean±SEM. from four separate experiments performed in duplicate. *, p<0.05 vs. normal control; #, p<0.05, ##, p<0.01 vs. palmitate-alone treated cells or islets.

Fig. 4

Kaempferol improves palmitate-induced dysfunction of INS-1E cells and human islets. INS-1E cells (A-C) and human islets (D-F) were incubated in RPMI1640 medium containing 0.5 mM palmitate (P) with or without 10 μM kaempferol (K) for 4 d. Cells were then washed and further cultured in KRB buffer containing either 3 (LG) or 20 mM glucose (HG) for 30 min at 37°C. Insulin secreted into KRB buffer (A and D) and inside the cells (B and E) was measured by an ELISA kit. The levels of PDX-1 protein in cell extracts was measured by immunoblot and normalized to β-actin content (C and F). Values are means ± SEM derived from four separate experiments performed in duplicate. *, p< 0.05 vs. normal control; #, p<0.05, ##, p<0.01 vs. palmitate-alone treated cells.

Fig. 5

kaempferol restores cAMP signaling impaired by palmitate in INS-1E β-cells. INS-1E cells were treated as stated in Fig. 1. (A) Intracellular cAMP levels were measured by EIA. (B) PKA activity in cell extracts was determined by measuring phosphorylation of fluorescent-labeled PKA substrate kemptide. (C) The phosphorylation of CREB (p-CREB) in the cells was detected by immunoblot analysis using a phospho-specific CREB antibody. The membrane was stripped and reprobed with a CREB antibody. (D) Cells were infected with adenoviral construct containing CRE-luc prior to above treatment. Firefly luciferase activity was measured with the dual reporter luciferase assay system and normalized to activity of the renilla control construct in the cell extracts. Values are mean ± SEM from three experiments performed in duplicate. *, p< 0.05 vs. normal control; #, p<0.05, ##, p<0.01 vs. palmitate-alone treated cells.

Fig. 6

Kaempferol prevents INS-1E β-cells from lipotoxicity-induced apoptosis and dysfunction via CREB. INS-1E cells transfected with scrambled siRNA (S) or siRNA directed against CREB were treated with 0.5 mM palmitate (P) or vehicle in the presence or absence of 10 μM kaempferol (K) for 4 d. The efficiency of the knockdown was determined by measuring total and phosphorylated CREB (p-CREB) protein levels in transfected cells (A and B). Cell apoptosis (C), GSIS (D), insulin content (E), and Bcl-2 protein expression (F) in the cells were determined. Values are mean ± SEM from four experiments performed in duplicate. *, p< 0.05 vs. normal control; #, p<0.05, ##, p<0.01 vs. palmitate-alone treated cells; and §, p<0.05 vs. P+K-treated cells.

Fig. 7

Kaempferol protects the cAMP signaling pathway in INS-1E β-cells exposed to palmitate via stimulation of PDX-1 expression. INS-1E cells were treated with kaempferol (K; 0.1, 1, 10 μM) for 48 h. Intracellular cAMP production was measured by EIA (A), and PDX-1 protein expression in cell extracts was determined by immunoblot analysis and normalized to β-actin content (B). INS-1E cells transfected with scrambled siRNA (S) or siRNA directed against PDX-1 (I) were treated with 10 μM kaempferol (K) for 48 h. siRNA-mediated knockdown of PDX-1 was verified by immunbot analysis of PDX-1 protein (C). Kaempferol-stimulated intracellular cAMP generation (D) and CREB phosphorylation (P-CREB) (E) in control and transfected cells were determined. Values are mean ± SEM from 4 experiments determined in duplicate. *, p< 0.05 vs. normal control; #, p<0.05 vs. kaempferol-alone treated cells.

Fig. 8

Kaempferol increases PDX-1 expression, maintains cAMP signaling, and subsequently prevents apoptosis of human islets after chronic exposure to palmitate. (A) Human islets treated with kaempferol (K; 1, 10 μM) for 48 h. PDX-1 protein expression in cell extracts was measured by immunoblot analysis and normalized to β-actin content. (B) Human islets were incubated with 0.5 mM palmitate (P) in the presence or absence of kaempferol (K; 0.1, 1, 10 μM) for 4 d, followed by measuring intracellular cAMP content. (C) Human islets were infected with 0, 25, 50 MOI MCREB virus/cell, or β-gal control virus. CREB phosphorylation in the cell lysates was measured with loading control monitored by measuring β-actin content. (D) Human islets were infected with 50 MOI MCREB virus/cell or the same number of control virus and then incubated with 0.5 mM palmitate (P) in the presence of 10 μM kaempferol (K) or vehicle for 4 d. Apoptosis of islet cells was measured as described in Fig. 2. Data are expressed as mean ± SEM obtained from three to four independent experiments measured in triplicate each. *, p< 0.05 vs. control; #, p<0.05, ##, p<0.01 vs. palmitate-alone treated cells.

Fig. 9

The stimulatory effect of kaempferol on PDX-1 expression may be structure-specific. INS-1E cells (A) and human islets (B) were cultured with various flavonoids (10 μM) in RPMI medium for 48 h. PDX-1 protein expression in cell extracts was measured by immunoblot analysis and normalized to β-actin content. (C) Chemical structures of tested compounds. Data are expressed as mean ± SE derived from three independent experiments. ##, P<0.01 vs. vehicle alone-treated cells. C: control, N: naringenin, H: hesperetin, E: epicatechin, G: epigallocatechin gallate, A: apigenin, L: luteolin, Q: quercetin, K: kaempferol.