Expression of glucose-dependent insulinotropic polypeptide in the zebrafish

Abstract

In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin (insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to be a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species.

Fig. 1.

Alignment of vertebrate incretin hormone sequences. A: human (NM_004123), rat (NM_019630), mouse (NM_008119), chicken (EF010535), frog (EF010533), and zebrafish (EF010535) glucose-dependent insulinotropic polypeptide (GIP) protein sequences were aligned using the ClustalW alignment program.

Fig. 2.

Analysis of GIP and insulin expression in zebrafish. A: zebrafish eggs/larvae were collected after timed matings. Total RNA was extracted, and cDNA was used as a template for PCR reactions to examine GIP (top), insa (middle), and β-actin (bottom) expression. B: zebrafish larvae were examined using whole mount in situ hybridization. Larvae were hybridized with insa antisense probe (a), insa sense probe (b), GIP antisense probe (c), or GIP sense probe (d). Red arrows indicate insa or GIP RNA expression. C: zebrafish adult tissues were assayed for GIP and β-actin expression. SI, small intestine; F, fat; L, liver; P, pancreas; H, heart; B, brain; T, tail. D: immunohistochemical analysis of adult zebrafish tissue. Intestinal sections were probed with goat anti-human insulin antiserum (a, e), rabbit anti-zebrafish GIP antiserum (b, c, f), or rabbit anti-human amylase antiserum (d). A merged image showing coexpression of GIP and insulin is shown in g. Yellow arrow in b indicates the location of GIP-positive cells. In c and d, green arrows in indicate amylase-positive cells and red arrows indicate GIP-positive cells. Purple arrows (e, f, g) indicate GIP-positive, insulin-negative cells. White arrows (e, f, g) indicate cells positive for both GIP and insulin. Nuclei (blue) were counterstained with DAPI. zfGIP, zebrafish GIP.

Fig. 3.

Analysis of rat GIP receptor (GIPR) and GLP-1R activation by zfGIP. A and B: GH3 cells were transfected with empty vector, zfGIP expression plasmid, or rat GIP expression plasmid. After 48 h, media were collected, and the ability of secreted GIP to activate GIPR (A) or GLP-1R (B) was assessed using specific bioassays. Activation of GIPR was expressed as fold activation over control (A) or raw activity (B). P values were calculated using Student's t-test. C–E: GH3 cells were plated on 48-well plates, incubated in DMEM without FBS overnight and pretreated with 1× IBMX to inhibit endogenous phosphodiesterase activity. Cells were then treated with 10–11 M to 10–9 M porcine GIP (C), 10–11 M to 10–9 M zfGIP (D), or 10–10 M to 10–9 M rat GLP-2 purified peptides (E) in the presence or absence of the GIPR antagonist ANTGIP. In addition, cells were treated with 10 μM forskolin (f) as a positive control, and untreated cells (u) were used as a negative control. Cell lysates were generated, and intracellular cAMP levels were measured using a competitive ELISA. P values were calculated using Student's t-test. **P < 0.05, ns = not significant.

Fig. 4.

Analysis of putative zebrafish glucagon (zh gluc) receptor and GLP-2R activation by zfGIP. GH3 cells were transfected with empty vector, zfGIP expression plasmid, or zf proglucagon II expression plasmid. After 48 h, media were collected, and the ability of secreted GIP and glucagon to activate zfGR (A) or zfGLP-2R (B) was assessed using specific bioassays. Activation of GIPR was expressed as fold activation over control (A) or raw activity (B). cont, Control. P values were calculated using Student's t-test.

Fig. 5.

Examination of food deprivation on GIP, proglucagon II, and insa expression levels. Larvae were collected from timed adult matings, and starting at 6 days postfertilization were fed a standard paramecium diet or had food withheld. Total RNA was isolated and analyzed by quantitative PCR for the expression of GIP (A), proglucagon II (B), and insa (C). mRNA levels were corrected for β-actin expression, and data were expressed as means ± SE or expression relative to expression at 28 h after feeding. P values were calculated using Student's t-test. *P < 0.05.