Glucose metabolism in the Belgrade rat, a model of iron-loading anemia

Abstract

The iron-diabetes hypothesis proposes an association between iron overload and glucose metabolism that is supported by a number of epidemiological studies. The prevalence of type 2 diabetes in patients with hereditary hemochromatosis and iron-loading thalassemia supports this hypothesis. The Belgrade rat carries a mutation in the iron transporter divalent metal transporter 1 (DMT1) resulting in iron-loading anemia. In this study, we characterized the glycometabolic status of the Belgrade rat. Belgrade rats displayed normal glycemic control. Insulin signaling and secretion were not impaired, and pancreatic tissue did not incur damage despite high levels of nonheme iron. These findings suggest that loss of DMT1 protects against oxidative damage to the pancreas and helps to maintain insulin sensitivity despite iron overload. Belgrade rats had lower body weight but increased food consumption compared with heterozygous littermates. The unexpected energy balance was associated with increased urinary glucose output. Increased urinary excretion of electrolytes, including iron, was also observed. Histopathological evidence suggests that altered renal function is secondary to changes in kidney morphology, including glomerulosclerosis. Thus, loss of DMT1 appears to protect the pancreas from injury but damages the integrity of kidney structure and function.

Keywords: iron overload; kidney.

Fig. 1.

Body weight (A) and daily food consumption per kg body wt (B) of Belgrade (b/b) and female heterozygote (+/b) rats. Data were recorded two times a week from 3 to 18 wk old. The food intake was adjusted by the body weight of each rat; n = 4 in both groups. *P < 0.05 vs. +/b in A at each time point. *P < 0.05 vs. +/b in B.

Fig. 2.

Glycometabolic status in +/b and b/b rats. Glucose tolerance test (GTT) (A) and insulin tolerance test (ITT) (B) were carried out after 6 h fasting. Twelve-week-old male b/b rats and age-matched +/b rats (n = 6 each group) were injected with d-glucose (1.5 g/kg body wt) or insulin (1 IU/kg body wt), and blood glucose was measured at 10, 20, 30, 45, 60, and 90 min after ip injection.

Fig. 3.

Insulin signaling in skeletal muscle of Belgrade rats. Phospho (p) Akt was determined by Western blot analysis (A), and the induced phosphorylation on Akt(Ser⁴⁷³) (S473) after insulin injection was compared with the total Akt level for b/b and +/b rats (n = 4 each group). Total Fe in skeletal muscle was measured by Inductively-Coupled Plasma Optical Emission Spectroscopy (B); n = 4 +/b and n = 3 b/b.

Fig. 4.

Pancreatic function in Belgrade rats. Hematoxylin and eosin staining of pancreas in +/b and b/b rats is shown (A). Glucose-induced insulin secretion was assessed (B). Insulin levels were determined in blood samples collected at 0, 10, 20, 30, and 45 min after glucose injection during GTT assay; n = 6 for both groups in A and B.

Fig. 5.

Pancreatic nonheme iron levels and pancreatic ferritin expression. Pancreatic nonheme iron levels in +/b and b/b are shown (A), and they are compared with pancreatic levels of control rats and carbonyl iron-loaded rats; n = 6 +/b and b/b, n = 3 in control and iron-loaded rats. Ferritin was determined by Western blot analysis in +/b, b/b, control, and carbonyl iron-fed rats (B and C), n = 3 in each group.

Fig. 6.

Kidney function and renal morphology in Belgrade rats. Urinary glucose excretion (A) was measured in 8- and 12-wk-old rats as described in materials and methods, n = 4 in both groups. Nonheme iron levels were determined in 12-wk-old rat kidneys (B), n = 4 in each group. Urinary K, Cl, Ca, and Fe were determined and adjusted to body weight (C). For K, Cl, and Ca, n = 3 each group. For Fe determinations, n = 6 +/b and n = 3 b/b. Periodic acid-Schiff (PAS) staining of paraformaldehyde-fixed sections demonstrated structural changes in the kidneys of b/b rats compared with +/b animals (D), n = 3 each group. The black arrows in DV show disorganization in renal cortex of b/b rats with an ill-defined corticomedullary junction, cellular infiltrations in renal tubules, and tubular dilatation. Arrows in DVI show occlusion of the luminal space in cortical tubules of b/b rats. The black arrows in D, VII and VIII, show thickening of glomerular basement membrane and an increased extracellular mesangial matrix. The yellow “g” in III and VII refers to the glomerulus.