Fatty diabetic lung: altered alveolar structure and surfactant protein expression

Abstract

Pulmonary dysfunction develops in type 2 diabetes mellitus (T2DM) in direct correlation with glycemia and is exacerbated by obesity; however, the associated structural derangement has not been quantified. We studied lungs from obese diabetic (fa/fa) male Zucker diabetic fatty (ZDF) rats at 4, 12, and 36 wk of age, before and after onset of T2DM, compared with lean nondiabetic (+/+) rats. Surfactant proteins A and C (SP-A and SP-C) immunoexpression in lung tissue was quantified at ages 14 and 18 wk, after the onset of T2DM. In fa/fa animals, lung volume was normal despite obesity. Numerous lipid droplets were visible within alveolar interstitium, lipofibroblasts, and macrophages, particularly in subpleural regions. Total triglyceride content was 136% higher. By 12 wk, septum volume was 21% higher, and alveolar duct volume was 36% lower. Capillary basement membrane was 29% thicker. Volume of lamellar bodies was 45% higher. By age 36 wk, volumes of interstitial collagen fibers, cells, and matrix were respectively 32, 25, and 80% higher, and capillary blood volume was 18% lower. ZDF rats exhibited a strain-specific increase in resistance of the air-blood diffusion barrier with age, which was exaggerated in fa/fa lungs compared with +/+ lungs. In fa/fa lungs, SP-A and SP-C expression were elevated at age 14-18 wk; the normal age-related increase in SP-A expression was accelerated, whereas SP-C expression declined with age. Thus lungs from obese T2DM animals develop many qualitatively similar changes as in type 1 diabetes mellitus but with extensive lipid deposition, altered alveolar type 2 cell ultrastructure, and surfactant protein expression patterns that suggest additive effects of hyperglycemia and lipotoxicity.

Fig. 1.

Representative micrographs of lean nondiabetic (+/+; top left) and obese diabetic (fa/fa) lungs (remaining panels) from 36-wk-old animals stained with Oil Red O. Top left: in +/+ lung, lipid deposits are absent (bar = 50 μm). Top right and bottom left: in fa/fa lung, numerous lipid deposits are visible within the septum and alveolar macrophages (bar = 50 μm); sections were counterstained with hematoxylin. Bottom right: the counterstain was omitted in a section from a fa/fa lung under lower magnification to demonstrate the preferential distribution of lipid deposits and alveolar macrophages near the pleural surface (bar = 100 μm).

Fig. 2.

Representative electron micrographs of +/+ and fa/fa lungs from 36-wk-old animals are shown. Top left: an alveolar type 2 (AT2) cell from +/+ lung is shown (bar = 5 μm). Top right and bottom left: in fa/fa lung, uncontrasted lipid droplets within a lipofibroblast (arrows) and irregular protein pools within AT2 cells (*) are shown (bar = 5 μm). Bottom right: interstitial lipid droplets (arrow) are contrast-enhanced by treatment with imidazole (bar = 1 μm).

Fig. 3.

Representative high-magnification electron micrographs from 36-wk-old animals show protein accumulation within dilated rough endoplasmic reticulum in fa/fa lungs but not in +/+ lungs. Bar = 1 μm.

Fig. 4.

Age-related changes in harmonic mean thickness of the diffusion barrier (τ_hb; top) and volumes of the alveolar septal interstitium (middle) and capillary blood (bottom) in fa/fa and +/+ animals. Means ± SD. *P < 0.05; †P = 0.07 fa/fa vs. corresponding +/+.

Fig. 5.

Triglyceride content in lean (+/+, n = 4; +/fa, n = 6) and fatty diabetic (fa/fa, n = 11) lungs and livers. Tissues were perfused clear of blood before assay. Means ± SD.

Fig. 6.

Surfactant protein A (SP-A) expression in wild-type (+/+, n = 7) and fatty diabetic (fa/fa, n = 6) lungs. Representative immunoblots of SP-A expression at age 14 and 18 wk are shown. Blots were reprobed for β-actin. Signal intensities at 38, 32, and 26 kDa were totaled, normalized to β-actin intensity, and expressed as percentage of the mean normalized total +/+ signal quantified on the same blot. Triplicate assays used separate lung tissue samples; results were averaged for individual animals. Means ± SD, unpaired t-test. *P < 0.05 fa/fa vs. corresponding +/+.

Fig. 7.

SP-C expression in +/+ (n = 5–7) and fa/fa (n = 6) lungs. Representative immunoblots at 14 and 18 wk are shown. Blots were reprobed for β-actin expression. Signal intensities of proteins at 21, 16, and 13 kDa were scored separately, normalized to β-actin, and expressed as percentage of the mean normalized +/+ signal for each protein quantified on the same blot. Triplicate assays used separate lung tissue samples; results were averaged for individual animals. Means ± SD, unpaired t-test. *P < 0.05 fa/fa vs. corresponding +/+.

Fig. 8.

SP-A protein expression during maturation in +/+ and fa/fa lungs. Representative immunoblots showing SP-A expression with increasing age. Total SP-A signal intensity per lane was normalized to the corresponding β-actin signal and expressed as percentage of the mean total normalized signal for 52-wk animals quantified on the same blot. Triplicate assays used separate lung tissue samples; results were averaged for individual animals. +/+ (n = 3, 5, 3, and 3) and fa/fa (n = 5, 5, 6, and 3) at 14, 18, 36, and 52 wk, respectively. Means ± SD, 1-way ANOVA: *P < 0.05 vs. 52 wk.

Fig. 9.

SP-C protein expression during maturation in +/+ and fa/fa lungs. Representative immunoblots show SP-C expression with increasing age. Signal intensities of proteins at 21, 16, and 13 kDa were scored separately, normalized to β-actin, and expressed as percentage of the mean normalized signal for 52-wk animals quantified on the same blot. Triplicate assays used separate lung tissue samples; results were averaged for individual animals. +/+ (n = 3, 5, 3, and 3) and fa/fa (n = 5, 5, 6, and 3) at 14, 18, 36, and 52 wk, respectively. Means ± SD, 1-way ANOVA: *P < 0.05 vs. 52 wk.