Presentations

Abstract

Introduction:

Insulin resistance typically precedes and ultimately leads to insufficient insulin production and the onset of type II diabetes. Using their non-human primate baboon model and the aromatase inhibitor letrozole, Pepe and colleagues have shown that baboon offspring born to mothers deprived of estradiol (E2) during the second half of gestation develop insulin resistance. Studies also showed that the number of microvessels and the microvessel/skeletal muscle fiber ratio, which is important for delivery of insulin and glucose to myofibers, as well as the size and amount of individual muscle fibers were significantly reduced in near term fetuses deprived of E2. Importantly, all parameters in fetuses and insulin resistance as well as microvessel number in offspring were restored to normal in animals treated with letrozole plus E2. Therefore, we proposed that the elevation in E2 during the second half of primate pregnancy promotes systemic micro-vascularization as well as growth of fetal muscle fibers essential for insulin sensitivity in adulthood. However, it remains to be determined whether the impairment of fetal skeletal muscle fiber growth in E2 deprived baboons is sustained in offspring.

Methods:

Animals Samples of vastus lateralis skeletal muscle, paraffin embedded were available from pre pubertal baboon offspring (n = 2-3/group) aged 3 years old born to mothers untreated or treated in utero with letrozole ± E2.

Immunohistochemistry Skeletal muscle sections (5 µm) were deparaffinized, treated for antigen retrieval and incubated overnight at 4C with human monoclonal anti-mouse antibody to slow myosin, washed and then incubated with peroxidase-conjugated goat anti-mouse secondary antibody and a Vector SG peroxidase substrate kit to stain the slow (Type I) fibers black. Sections were washed and then incubated with an alkaline phosphatase-conjugated rabbit monoclonal anti-mouse antibody to fast myosin. After washing, sections were incubated with Vector red alkaline phosphatase substrate solution to stain the fast (Type II) fibers red, washed and cover-slipped with Xylene-based Cytoseal XYL.

Image Analysis Slides were imaged using an Olympus BX41 fluorescent microscope fitted with a DP70 camera and associated software (Olympus America, Inc.). Initial studies were performed in sections of skeletal muscle from 3 offspring untreated and 2 treated with letrozole and 2 with letrozole ± E2. Approximately 10-30 slow fibers and 25-70 fast fibers within randomly selected regions of myofibrils in each baboon were analyzed using Image J software (NIH). The number and size of each individual fast and slow fiber, the total area occupied by slow and fast fibers was determined in each of the regions analyzed and an overall mean calculated for each animal.

Results:

In the 3 groups of offspring, the number of fast fibers was 3-4 fold greater than the number of slow fibers but fiber numbers were not altered by treatment with letrozole. However, size (µm2) of slow (mean ± SE; 2,004 ± 402) and fast fibers (3,696 ± 542) appeared to be reduced in letrozole treated offspring (553 and 901 slow; 1,084 and 1,600 fast) and restored in one of the 2 offspring treated with letrozole + E2 (750 and 4,262 slow; 1,261 and 5,600 fast). Thus, the area of myofibers (fiber number x size) comprised of fast fibers exceeded area comprised of slow fibers. However, the area of slow and perhaps fast fibers in untreated offspring (33,875 ± 18,187 slow; 187,875 ± 44,490 fast) appeared to be reduced in letrozole treated offspring (5,530 and 9,911 slow; 42,276 and 144,000 fast) and restored in one offspring treated with letrozole + E2 (63,930 and 10,500 slow; 156,800 and 89,531 fast).

Conclusions:

These very preliminary results which require a more robust analysis using more sections and additional animals indicates that the decrease in slow and fast fiber size and area characteristic of fetal skeletal muscle of animals deprived of estrogen in utero is still apparent in skeletal muscle of prepubertal offspring deprived of estrogen in utero. These findings which are very preliminary and based on a small sampling of tissue regions are nonetheless supportive of the suggestion that impaired skeletal muscle growth seen in the fetus is sustained prior to the onset of puberty in offspring deprived of estrogen in utero. It also remains to be determined whether these preliminary findings are sustained after puberty and induction of secretion of gonadal hormones estrogen (females) or testosterone (males).