Kim L. Hossner

Satellite cell regulation following myotrauma caused by resistance exercise.

It is generally accepted that the primary mechanisms governing skeletal muscle hypertrophy are satellite cell activation, proliferation, and differentiation. Specific growth factors and hormones modulate satellite cell activity during normal muscle growth, but as a consequence of resistance exercise additional regulators may stimulate satellite cells to contribute to gains in myofiber size and number. Present knowledge of the regulation of the cellular, biochemical and molecular events accompanying skeletal muscle hypertrophy after resistance exercise is incomplete. We propose that resistance exercise may induce satellite cells to become responsive to cytokines from the immune system and to circulating hormones and growth factors. The purpose of this paper is to review the role of satellite cells and growth factors in skeletal muscle hypertrophy that follows resistance exercise.

Insulin-like growth factors and their binding proteins in domestic animals

Insulin-like growth factors (IGFs) and their binding proteins play an essential role in regulating animal growth and metabolism. The initial portion of the current review focuses on the physiological effects of the IGFs and delineates their role as regulators of animal growth and metabolism. The role of IGFs as mediators of growth hormone effects, as insulin-like metabolic regulators and as foetal growth regulators is discussed. The remainder of the review is devoted to the IGF binding proteins, their modulation of IGF action and their role in foetal and postnatal regulation of growth.

The development and utility of a defined muscle and fat co-culture system

The utility of co-culture systems in defining the interactions between two different cell types has been well documented in the literature but is a relatively new tool for use in the study of cells derived from normal muscle tissue from meat animals. The majority of myonuclei in postnatal skeletal muscle cells (myofibers) result from satellite cell proliferation, differentiation and fusion with existing myofibers. As such, satellite cell culture systems that mimic postnatal myofibers have great potential for delineating the process of growth and repair of muscle mass. Other ways to simulate the environment of postnatal myofibers might include the development of co-culture systems using satellite cells, or satellite cell-derived myotubes, and other mesodermal cells commonly found associated with muscle tissue in vivo. This brief review describes our initial efforts to develop a defined satellite cell and preadipocyte co-culture system. We provide useful information about defined media requirements and requirements for proper cell orientation and growth on two different growth planes. We present summary data to suggest that differences were found between members of the insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) families of polypeptides, when conditioned media samples were analyzed from co-cultures composed of 3T3-L1 preadipocytes and satellite cells (with different propensities to undergo morphological fusion to form multinucleated myotubes). We also provide information about potential problems to avoid when initiating and conducting co-culture experiments. Such a co-culture system has application in the study of human obesity and also in the regulation of fat deposition in meat animals.

Comparison of immunochemical (enzyme-linked immunosorbent assay) and immunohistochemical methods for the detection of central nervous system tissue in meat products

Three methods are widely used in the United States to detect the presence of central nervous system (CNS) tissue in meat products: the fluorescent enzyme-linked immunosorbent assay (F-ELISA), developed in this laboratory, the colorimetric Ridascreen Risk Material 10/5 ELISA (R-ELISA), and the U.S. Department of Agriculture, Food Safety and Inspection Service immunohistochemical (IHC) procedure. These assays are based on the immunological detection of glial fibrillary acidic protein (GFAP), a neural antigen largely restricted to the CNS. The objective of the current study was to compare the sensitivity and repeatability of these tests for detecting the presence of neural tissue in meat. Ground beef spiked with 0.05 to 0.5% of bovine brain, spinal cord (SC), or dorsal root ganglia, as well as advanced meat recovery samples, were evaluated by each of the three GFAP detection procedures. Interassay coefficients of variation for the F-ELISA GFAP standards were 7 to 25%, and intra-assay variation due to sampling and extraction of spiked ground beef was 7 to 13% for SC and 8 to 14% for brain (n = 10). The F-ELISA was the most sensitive of the methods tested, capable of detecting 0.3 ng GFAP standard per well and the presence of 0.05% brain and SC in meat. The R-ELISA standards produced highly variable results (up to 36% variation) and, as a result, none of these standards were different from zero (n = 26). The R-ELISA resulted in high sample variation in SC-spiked ground beef samples (coefficients of variation were 23 to 50%) and did not detect the presence of brain contamination. After modification of the R-ELISA sampling and extraction methods, SC-spiked sample variation was reduced to 16 to 20%, and sensitivity was improved from 0.3 to 0.2% SC, although brain tissue was still not detected. The IHC analysis of CNS-adulterated ground beef had a sensitivity of 0.2% SC and 0.05% brain, with false-negative rates of 10 to 20% at and above the stated sensitivities. None of the methods examined detected dorsal root ganglia contamination. The F-ELISA detected the presence of CNS contamination in 20% of the advanced meat recovery samples, compared to 3.5 to 5% for the R-ELISA and 2% for IHC. This study suggests that the F-ELISA is much more sensitive and repeatable than either the R-ELISA or the IHC procedure method for the detection of CNS tissue in meat products.

Metabolic rate, organ mass, and mitochondrial proton leak variations in lean and obese rats

The purpose of this study was to determine if differences in metabolic rate between lean and obese strains of rats were associated with differences in proton leak across the inner mitochondrial membrane. Metabolic rates were determined for each of five obese Zucker, Sprague-Dawley, and Fisher 344 rats and three lean Zucker rats by 24-hour indirect respiration calorimetry measurements. Feed intakes were different (P < 0.05) among all strains, with the obese Zucker rats having the greatest intakes. Adjusted to a common dietary intake, the obese Zucker rats had at least 21% lower heat productions than the lean strains of rats. Following the calorimetry measurements, the rats were sacrificed, internal organs were removed and weighed and mitochondria were isolated from the liver. Internal organs composed a larger proportion of lean body mass in obese compared to lean rats. Respiration rates and membrane potentials of the mitochondria were then determined. Proton leak kinetics were visualized by plotting proton leak (calculated from respiration rate) against membrane potential. The lean rats had a 2-3-fold higher proton leak rate than the obese Zucker rats at the same membrane potential. A low mitochondrial proton leak rate may explain part of the abnormal heat productions and bioenergetic efficiencies in the obese Zucker rat.

INSULIN-LIKE GROWTH FACTOR (IGF)-I AND -II AND IGFBP SECRETION BY OVINE SATELLITE CELL STRAINS GROWN ALONE OR IN COCULTURE WITH 3T3-L1 PREADIPOCYTES

SummaryThe current study was designed to examine the effects of muscle and fat stem cell coculture on the secretion of insulinlike growth factor (IGF)-I and -II and IGF binding proteins (IGFBP) by these cells. Two sheep satellite cell strains with negligible or high potential for differentiation (10A and 01, respectively) were placed in coculture with 3T3-L1 preadipocytes using a filter support to separate the two cell types. Media conditioned by the cells grown alone or in coculture were analyzed for IGFs by RIA or IGFBPs by ligand blotting. The numbers of satellite cells and preadipocytes declined throughout the 5-d culture period, although coculture slowed the 3T3-L1 decline but hastened the satellite cell decline. The satellite cell strains and 3T3-L1 cells secreted small amounts of IGF-I (≤2 ng/ml) and IGF-II (<10 ng/ml) over the 5-d culture period. Coculture did not increase the amount of IGF-I and -II in conditioned media. The lowly differentiating 10A cells secreted barely detectable amounts of the low molecular weight IGFBP-3 subunit (34 kDa), IGFBP-2 (28 kDa), and IGFBP-4 (18 kDa). Coculture of 10A and 3T3-L1 cells potentiated secretion of IGFBP-2 and-3. Strain 01, which readily differentiates, secreted high levels of both IGFBP-3 subunits (34 and 39 kDa) and IGFBP-2 (28 kDa), as well as significant amounts of the 18 kDa IGFBP-4. Coculture did not alter IGFBP secretion of 01 cells. This study showed that while IGF-I and -II levels in media conditioned by sheep satellite cell strains are low and relatively invariant, the intensity and complexity of IGFBP patterns increases with time in culture and with the potential for differentiation of the satellite cell strains. Coculture with preadipocytes appeared to potentiate IGFBP secretion while reducing satellite cell viability.

Detection of central nervous system tissue on meat and carcass-splitting band saw blade surfaces using modified fluorescent glial fibrillary acidic protein enzyme-linked immunosorbent assay sampling and extraction procedures.

This study was conducted to determine optimal buffer pH, extraction procedure, and temperature for detecting central nervous system (CNS) tissue on meat surfaces and on carcass-splitting band saw blades using swab sampling. Glial fibrillary acidic protein (GFAP) is restricted to CNS tissue and has been used as a marker for CNS tissue presence in meat products. Sample preparation, extraction procedure, and extraction temperature of glial fibrillary acidic protein fluorescent enzyme-linked immunosorbent assay (GFAP F-ELISA) were modified to detect CNS tissue on meat surfaces and on carcass-splitting band saw blades. Maximum GFAP recovery was observed with an extraction buffer pH of 7.4. Extracting samples at room temperature by vortexing for 30 s in 1 ml of extraction buffer (phosphate-buffered saline [pH 7.4] plus 0.05% sodium dodecyl sulfate) consistently provided detection of GFAP on meat surfaces contaminated with 500 microg of spinal cord suspension per 50 cm2 and on carcass-splitting band saw blades contaminated with 20 microg of spinal cord suspension per 50 cm2. Recovery of GFAP was not affected by storing samples overnight at 4 degrees C. The current studies demonstrate the effectiveness of modified sampling procedures and preparations, sample extraction buffer pH, and extraction temperatures. These modifications introduced to the original F-ELISA sampling protocol result in asensitive and repeatable assay for detection of CNS tissue on meat surfaces and on carcass-splitting band saw blades.

Effects of bovine colostral ultrafiltrates on growth and differentiation of 3T3-L1 preadipocytes

This study was designed to compare the effects of whole and size‐fractionated bovine colostrum with bovine calf serum (BCS) on the growth and differentiation of 3T3‐L1 fibroblasts. High (HMW) and low (LMW)‐molecular‐mass ultrafiltrate fractions of colostrum were prepared from defatted colostrum (COL) by diafiltration through membranes with a molecular‐mass cut‐off of 30 kDa. Incorporation of [3H]thymidine into the cells was used as a reflection of DNA synthesis/cell proliferation. The growth‐promoting activity of LMW was 2.3‐ and 2.5‐fold higher than COL and HMW, respectively (P<0.05), and 185 μg/ml LMW stimulated cell proliferation equivalent to 10% BCS. Although insulin‐like growth factor (IGF)‐I, IGF‐II and platelet‐derived growth factor AB stimulated 3T3‐L1 cells, antibodies to these factors did not inhibit the LMW effects. The LMW fraction was about twice as effective as COL and HMW in stimulating differentiation of the cells into adipocytes, but maximal differentiation was only 60% of that seen with 10% fetal bovine serum (FBS). Treatment with COL, HMW, IGF‐I and insulin induced peroxisome‐proliferator‐activated receptor γ RNA, but levels were about half of that with 10% FBS treatment and LMW induction was 80% of FBS. Low amounts of leptin mRNA were detected in adipocytes and abundance did not differ between treatments with BCS, hormones or COL fractions. This study showed that bovine colostral LMW stimulated the growth and differentiation of 3T3‐L1 preadipocytes and may be a useful serum substitute to support the growth of these cells.