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. 2015 Apr 15;308(8):E662-9.
doi: 10.1152/ajpendo.00441.2014. Epub 2015 Feb 10.

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Lisette A Maddison  1 , Kaitlin E Joest  1 , Ryan M Kammeyer  2 , Wenbiao Chen  3
Affiliations

Skeletal muscle insulin resistance in zebrafish induces alterations in β-cell number and glucose tolerance in an age- and diet-dependent manner

Lisette A Maddison et al. Am J Physiol Endocrinol Metab. .

Abstract

Insulin resistance creates an environment that promotes β-cell failure and development of diabetes. Understanding the events that lead from insulin resistance to diabetes is necessary for development of effective preventional and interventional strategies, and model systems that reflect the pathophysiology of disease progression are an important component toward this end. We have confirmed that insulin enhances glucose uptake in zebrafish skeletal muscle and have developed a zebrafish model of skeletal muscle insulin resistance using a dominant-negative IGF-IR. These zebrafish exhibit blunted insulin signaling and glucose uptake in the skeletal muscle, confirming insulin resistance. In young animals, we observed an increase in the number of β-cells and normal glucose tolerance that was indicative of compensation for insulin resistance. In older animals, the β-cell mass was reduced to that of control with the appearance of impaired glucose clearance but no elevation in fasting blood glucose. Combined with overnutrition, the insulin-resistant animals have an increased fasting blood glucose compared with the control animals, demonstrating that the β-cells in the insulin-resistant fish are in a vulnerable state. The relatively slow progression from insulin resistance to glucose intolerance in this model system has the potential in the future to test cooperating genes or metabolic conditions that may accelerate the development of diabetes and provide new therapeutic targets.

Keywords: diabetes; glucose tolerance; zebrafish; β-cell.

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Figures

Fig. 1.
Fig. 1.
Impaired insulin signaling in zMIR (zebrafish muscle insulin resistance) fish. A: expression of the dominant-negative IGF-I receptor (IGF-IR) indicated by enhanced green fluorescent protein (EGFP) fluorescence in a live 3-mo-old fish. B: longitudinal section through the trunk of a 3-mo-old fish highlighting GFP immunostaining (brown) with hematoxylin counterstaining. C: GFP immunofluorscence of highlighted individual muscle bundles. D and E: GFP immunofluorscence in zMIR (D) or nontransgenic (E) fish. GFP is present in skeletal muscle (m) but has no specific signal in liver (li) or intestine (i). F: Western blot for phosphorylated and total Akt in skeletal muscle samples of fasted fish after both feeding and insulin injection. G: analysis of Akt phosphorylation compared with total Akt levels in fasted animals. No difference in basal phosphorylation is observed. H: quantification of Akt phosphorylation relative to total Akt levels in the 3 treatment conditions. Level of Akt phosphorylation in fasted control animals was set to 1. A significant increase in Akt phosphorylation was observed in the control animals with each treatment, but no increase in phosphorylation was observed in the zMIR animals; n = 8–10 animals/group. I: uptake of 2-deoxyglucose (2-DG) into skeletal muscle with and without coinjection of insulin. Level of 2-DG was set to 1 for the control glucose-injected group. A significant increase in 2-DG uptake was seen in control animals with coinjection of insulin but no change in the zMIR animals; n = 8–13 animals/group. All values are means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 2.
Fig. 2.
Progressive glucose intolerance in zMIR fish. A: intraperitoneal (ip) glucose tolerance test in 3-mo-old zMIR and control fish with blood glucose levels determined after fasting (0 min) and 30, 90, and 180 min after injection of 0.5 mg glucose/g body wt; n = 10–14 fish/time point. No difference in glucose tolerance between genotypes was observed. B: fasting blood glucose in 3-mo-old fish. The increase in fasting blood glucose was not statistically significant; n = 10 fish/genotype. C: ip glucose tolerance test in 1-yr-old zMIR and control fish following injection of 0.5 mg glucose/g body wt; n = 8–13 fish/time point. D: fasting blood glucose in 1-yr-old fish. The increase in fasting blood glucose was not statistically significant; n = 47 fish/genotype. All values are means ± SE. *P < 0.05.
Fig. 3.
Fig. 3.
Increased no. of β-cells in young zMIR fish. A: quantification of the no. of β-cells during juvenile growth of zMIR (open bars) and control (gray bars) siblings. The no. of β-cells at 7 (n = 18 and 17 zMIR and controls, respectively), 14 (n = 87 and 84), and 21 days postfertilization (dpf) (n = 48 and 47) was not statistically different. At 28 dpf (n = 46 and 52), there was a statistically significant increase in the no. of β-cells in the zMIR animals (P < 0.001, t-test). B and C: principal islet of 28 dpf control (B) and zMIR (C) fish using Tg(−1.2ins:H2BmCherry) to label the β-cells. Scale bars, 20 μm. D and E: examples of pancreatic tissue from 3-mo-old control (D) and zMIR (E) animals. The nuclei of β-cells were labeled by Tg(−1.2ins:H2BmCherry), and the margins of the pancreatic tissue are indicated by the dotted line. Scale bars, 100 μm. F: quantification of β-cells in 3-mo-old fish (n = 10 control, 22 zMIR). The no. of β-cells is normalized to the total pancreatic tissue. A significant increase in β-cells was observed in the zMIR fish (P < 0.01, t-test). G: quantification of β-cells in 1-yr-old fish. No significant difference between genotypes was observed. H and I: 5-ethynyl-2-deoxyuridine (EdU) labeling of β-cells in 28 dpf control (H) and zMIR (I) fish. Each image is a single slice of a confocal stack. Arrows indicate EdU-positive β-cells. Scale bars, 20 μm. J: quantification of β-cell proliferation. An increase in β-cell proliferation was observed in zMIR fish in both the principal and secondary islets; n = 12 for each genotype. All values are means ± SE. *P < 0.05; **P < 0.01.
Fig. 4.
Fig. 4.
Increased insulin mRNA in zMIR fish. RNA was isolated from the gastrointestinal tract or isolated pancreas of fasted 10-wk-old control and zMIR fish. The transcript levels for insulin (A) and glucagon (B) were compared with amylase as a marker for the endocrine pancreas. A statistically significant increase in the insulin transcripts (***P < 0.001, t-test), as well as a significant increase in glucagon mRNA (*P < 0.05, t-test), was observed; n = 10 fish for each genotype.
Fig. 5.
Fig. 5.
Overfeeding increases fasting blood glucose in zMIR fish. A: weight gain. For the 1st 2 wk, fish were maintained on a normal feeding schedule, and no change in body weight was observed. For the 2nd 2 wk, feedings were increased 5- to 10-fold, which induced an increase in body weight. No difference between genotypes was observed. B: weight gain in males and females. Female fish gained more weight than male fish on the overfeeding protocol, but no difference between genotypes was observed. C: fasting blood glucose. An increase in fasting blood glucose was observed with the increased feeding and body weight. After the 1st week of overfeeding there was no difference between genotypes, but in the 2nd week the zMIR fish had a significant elevation of fasting blood glucose compared with controls. All values are means ± SE. **P < 0.01.
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