Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices.

Elucidation of mechanisms underlying collagen fibril assembly and matrix-induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in-vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid-solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half-times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary-like network formation. This work documents the importance of oligomers as another physiologically-relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications.

Cloning and expression of porcine adiponectin, and its relationship to adiposity, lipogenesis and the acute phase response.

Adiponectin is an adipocyte-derived hormone that has been implicated recently in the regulation of inflammation in immunocytes, and in lipid metabolism and glucose homeostasis in liver, skeletal muscle and adipocytes. However, information in non-rodent models is limited. We have cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin in vivo following lipopolysaccharide (LPS) or E. coli administration. The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin respectively, and 79-83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species. Relative serum adiponectin concentrations were not altered in pigs infused with E. coli, and mRNA expression in adipose tissue was not responsive to LPS. However, analysis of serum from very lean vs a substantially fatter genotype of pig indicated that relative circulating adiponectin concentrations are higher (P<0.01) in the lean pigs than in the fatter genotype, and that the difference is established relatively early in the growth curve. Also, incubating pig adipocytes for 6 h with recombinant pig adiponectin resulted in an approximately 30% reduction (P<0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin. This is the first report in any species that adiponectin antagonizes the incorporation of glucose carbon into lipid in the adipocyte, and provides additional evidence that adiponectin acts as an autocrine regulatory factor to regulate energy metabolism.

Nutritionally induced adipose hypertrophy in young pigs is transient and independent of changes in the expression of the obese and peroxisome proliferator activated receptor genes.

Previous studies have shown that piglets weaned to a liquid milk replacer (MR), rather than a typical dry diet (DD) regimen, have improved growth rates and deposit more energy as body fat. In the present study, we used this model to determine whether changes in the expression of genes linked to the regulation of adiposity were related to the accelerated fat accretion. We also determined whether the increase in body fat was sustained throughout a substantial proportion of the growth curve. At weaning (19 plus minus 2 days of age), 96 piglets were placed in 12 replicate pens per diet (4 pigs per pen, 2 barrows and 2 gilts), and fed a liquid MR or conventional DD regimen for 5 weeks. Thereafter, 6 barrows and 6 gilts pigs from each diet were killed for determination of whole body chemical composition (less gastrointestinal contents). The remaining pigs were assigned randomly to weight target groups (60, 85, and 110 kg), placed in individual pens, and fed a conventional dietary regimen until killed at their respective weight targets for tissue sampling and determination of whole body chemical composition. Over the 5-week period in which the MR was fed, the growth rate of the pigs consuming the MR exceeded that of the pigs fed the DD by 36% (P <.05). Fat gain in these pigs was increased to 1.8 times that of the pigs fed the DD, and percentage body fat was 45% greater (P <.05). Acetyl Co-A carboxylase (ACC) activity (per mg of adipose extract protein) was not different between the two diet groups at the conclusion of the 5-week period, or at 110 kg body weight. During the MR period, actual protein gain was increased (P <.05) 22% in the pigs fed the MR as well. By 110 kg of body weight, body fat was reduced (P <.05) by 7.7% (total fat mass) and 8.3% (percentage of body weight basis) in the pigs fed MR vs. the DD group. The expression of the peroxisome proliferator activated receptors (PPAR) alpha and gamma was not influenced by diet or by body weight. Expression of the obese gene was independent of diet, but was greater (P <.09) in pigs at 110 kg body weight than at 60 kg. These data provide additional evidence that piglets weaned to liquid diets have greater rates of growth and deposit more body fat, but that this difference subsides quickly when a typical dry dietary regimen is imposed. Furthermore, the biochemical changes responsible for the increased adiposity are independent of changes in the expression of the obese or PPAR genes, at least at the mRNA level.

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1).

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.

Effect of dietary energy source and immunological challenge on growth performance and immunological variables in growing pigs.

Forty-eight growing pigs (23 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial. Dietary energy source (conventional [CON] vs high-oil corn [HOC]), with or without an immunological challenge (IC) regimen constituted main effects. The IC regimen consisted of injection of endotoxin (E. coli lipopolysaccharide [LPS]) and vaccination for porcine respiratory and reproductive syndrome (PRRS). Growth performance data were collected over a 5-wk period and are presented as prechallenge (d 1 to 14; d 1 was the 1st d of the study), challenge (d 15 to 21), and postchallenge (d 22 to 36) periods, and overall. Overall, the pigs fed HOC consumed less feed (P < .11) and gained more efficiently (P < .03). During the immunological challenge period, ADG was depressed 21% and feed intake 15% (P < .01). The IC resulted in lower (P < .01) serum alpha-1-acid glycoprotein (AGP) concentrations on d 22, and the magnitude of the reduction was greater in the pigs fed the CON diet (energy source x immune challenge, P < .10). Serum AGP concentrations remained lower (P < .08) in challenged pigs on d 36. Immunoreactive prostaglandin concentrations were higher (55%, P < .08) in the pigs fed HOC immediately following the IC period (d 22). The data reported herein indicate that the performance of pigs fed HOC is satisfactory, and that feeding HOC does not compromise growth performance during or after an immunological challenge.