Diabetes is growing public health problem in the United States. Both in Type 1 and Type 2 diabetes, the deterioration of glycemic control over time is largely due to insulin secretory dysfunction and significant loss of functional β-cells. As such, the search for novel agents that promote β-cell survival and preserve functional β-cell mass are one of the essential strategies to prevent and treat the onset of diabetes. Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. It was recently shown that dietary intake of foods containing genistein improves diabetes in both experimental animals and humans. However, the potential anti-diabetic mechanisms of genistein are unclear. In the present study, we first investigated the effect of genistein on β-cell insulin secretion and proliferation and cellular signaling related to these effects in vitro and in vivo. We then determined its anti-diabetic potential in insulin-deficient and obese diabetic mouse models. The results in our study showed that exposure of clonal insulin secreting (INS1E) cells or isolated pancreatic islets to genistein at physiologically relevant concentrations (1-10 μM) enhanced glucose-stimulated insulin secretion (GSIS), whereas insulin content was not altered, suggesting that genistein-enhanced GSIS is not due to a modulation of insulin synthesis. This genistein's effect is protein tyrosine kinase- and KATP channel-independent. In addition, genistein had no effect on glucose transporter-2 expression or cellular ATP production, but similarly augmented pyruvate-stimulated insulin secretion in INS1E cells, indicating that genistein improvement of insulin secretion in β-cells is not related to an alternation in glucose uptake or the glycolytic pathway. Further, genistein (1-10 μM) induced both INS1 and human islet β-cell proliferation following 24 h of incubation, with 5 μM genistein inducing a maximal 27% increase. The effect of genistein on β-cell proliferation was neither dependent on estrogen receptors, nor shared by 17β-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of PKA or ERK1/2 completely abolished genistein-stimulated β-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for β-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in both insulin deficient type 1 and obese type 2 diabetic mice, concomitant with improved islet β-cell proliferation, survival, and mass. These changes were not due to alternations in animal body weight gain, food intake, fat deposit, plasma lipid profile, or peripheral insulin sensitivity. Collectively, these findings provide better understanding of the mechanism underlying the anti-diabetic effects of genistein. Loss of functional β-cell mass through apoptosis is central to the development of both T1D and T2D and islet β-cell preservation and regeneration are very important components of β-cell adaptation to increased apoptosis and insulin resistance and therefore holds promise as a treatment for this disease. In this context, these findings may potentially lead to the development of novel low-cost natural agents for prevention and treatment of diabetes. / Ph. D.

A detailed study was made in this doctoral thesis on the development and differentiation of the vertebrate pituitary gland, with the aim to establish a fate map in Rathke's pouch of the origin of different hormone producing cells present in the adult pituitary gland, that explain if the differences observed in the distribution pattern of different hormone producing cells in the adult is the consecuence of differences in their development. For this reason, the study was made in two vertebrate groups, Mammals and Avian, that present notable differences in their hormone producing cell distribution patterns. The results allowed us to conclude that the origin of different hormone producing cells in Rathke’s pouch determine their definitive distribution in the adult gland. At the same time, the relationship between proliferation and differentiation was studied, showing us that after differentiation, hormone producing cells continue proliferating with a low rate, contributing to the establishment of differentiated populations. Using immunochemicals and in situ hidridization techniques, the expression of different molecules such as hypothalamic releasing factors; different peptides, whose role as modulators in different pituitary axis have been proposed in the adult animal; different calcium binding proteins and transcription factors in relation to the differentiation of different hormone producing cells, was also studied in this work, allowing us to establish different relationships between some of these factors and specific aspects of the development and differentiatin of the pituitary gland.

No / Bone morphogenetic protein (BMP) signaling plays a key role in the control of skin development and postnatal remodeling by regulating keratinocyte proliferation, differentiation, and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 (transgenic mice overexpressing a constitutively active form of Smad1 under K14 promoter) mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and quantitative real-time reverse-transcriptase-PCR (qRT-PCR) analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myosin VA (Myo5a), in the epidermis of K14-caSmad1 mice versus wild-type (WT) controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared with WT controls. Finally, small interfering RNA (siRNA)-mediated silencing of BMPR-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17, and Myo5a compared with controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization, and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

<p dir="ltr">The pancreas is composed of a variety of cell types such as acinar, endocrine, and ductal cells, as well as endothelial cells and adipocytes. Whereas we understand the distinct functions of each, there remains an incomplete understanding of the molecular pathways and communications that exist between these cells that may influence development, growth, and function. Given that diabetes is characterized by the destruction or dysfunction of the insulin-producing pancreatic beta cell, a better understanding of the mechanisms that influence cell growth and maintenance in the pancreas is of therapeutic interest. Genome-wide association studies identified eukaryotic initiation factor 5A (eIF5A) to be within a type 1 diabetes susceptibility locus, which also suggests this translation factor may play a role in maintaining beta cell health. EIF5A is active once post-translationally modified by the rate-limiting enzyme deoxyhypusine synthase (DHPS) in a process known as hypusination, producing hypusinated eIF5A (eIF5A^HYP). The functional loss of eIF5A^HYP via pancreas-specific genetic deletion of <i>Dhps</i> or <i>Eif5a</i> within multipotent pancreatic progenitor cells (MPPCs) results in an mRNA translation defect detectable at E14.5 causing the decreased expression of many proteins required for exocrine growth and function. Moreover, DHPS^ΔPANCmice die by 6 weeks-of-age; however, eIF5A^ΔPANCmice survive up to 2 years-of-age. The postnatal phenotype of the eIF5A^Δ^PANCmodel was investigated in this thesis.</p>

Yes / Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically. / This work was supported by the British Heart Foundation.

Steroid hormones are reported to increase oxytocin (OXT) expression in skeletal muscle. It is possible that OXT may play essential roles in satellite cell (SC) proliferation and differentiation, which further contribute to skeletal muscle development and growth. In this dissertation, we tested this hypothesis with in-vivo and in-vitro studies in intrauterine growth restriction (IUGR) sheep, caloric restricted (CR) calves, tamoxifen (TAM) treated heifers, and bovine satellite cells (BSCs), respectively. In the in-vivo studies, we collected (1) longissimus dorsi muscle (LM) from IUGR sheep; (2) infraspinatus muscle (INF), LM, and semitendinosus muscle (ST) from CR calves; (3) LM muscle from TAM heifers. In all samples, mRNA abundance of targeted genes, such as OXT, were measured. Muscle fiber size and BSC number were also determined in skeletal muscle from TAM treated heifers. For the in-vitro studies, different treatments including 17β-estradiol (E2), trenbolone (TBA), TAM, OXT, and atosiban were applied to wild-type BSC and OXT knockout BSC (CRISPR-OXT) separately to investigate OXT's functions on BSC activity. For in-vivo studies, OXT expression significantly decreased (P<0.05) in IUGR LM muscle. Caloric restriction decreased OXT expression (P<0.05) in INF, LD, and ST muscle. Expression of OXT in LM from TAM animals decreased 50% (P<0.05). Moreover, TAM caused a small statistical reduction (P<0.05) in cross-sectional area (CSA). In in-vitro studies, OXT alone increased (P<0.05) fusion index but not proliferation in the wild-type BSC, whereas both proliferation and differentiation were stimulated (P<0.05) by OXT treatment in the CRISPR-OXT cell. By contrast, E2 and TBA increased (P<0.05) both proliferation rate and fusion index in wild-type BSC. However, E2 and TBA only stimulated proliferation rate (P<0.05) but not fusion index for CRISPR-OXT cells. Atosiban treatment resulted in lower proliferation and differentiation (P<0.05) in both wild-type BSC and CRISPR-OXT cell compared with OXT and E2 treatment groups. Together, our in-vivo studies indicate that OXT may play important roles in skeletal muscle development and growth. Our in-vitro studies demonstrate that OXT plays important roles in BSC proliferation and differentiation, and it is involved in steroid hormone stimulated BSC activity. / PHD / Oxytocin (OXT) is a mammalian neurohypophysial hormone. It has been demonstrated that OXT is highly expressed in skeletal muscle and satellite cell (SC) by steroid hormone administration. However, the role of OXT in SC proliferation and differentiation is not elucidated. In this dissertation, the in-vivo and in-vitro studies are combined and used to investigate if OXT is involved in bovine SC (BSC) activity. In the in-vivo studies, we collected muscle samples from intrauterine growth restriction (IUGR) sheep, caloric restricted (CR) calves, and tamoxifen (TAM) treated heifers. In all samples, mRNA abundance of OXT was measured. For the in-vitro studies, wild-type BSC and OXT knockout BSC (CRISPR-OXT) were treated with different factors including 17β-estradiol (E2), trenbolone (TBA), TAM, OXT, and atosiban separately to investigate OXT's functions on BSC activity. For in-vivo studies, OXT expression significantly decreased (P<0.05) in IUGR, CR, and TAM muscle. In in-vitro studies, OXT alone increased (P<0.05) fusion index but not proliferation in the wild-type BSC, whereas both proliferation and differentiation were stimulated (P<0.05) by OXT treatment in the CRISPR-OXT cell. By contrast, E2 and TBA, which can stimulate OXT expression in cultured BSC, increased (P<0.05) both proliferation rate and fusion index in wild-type BSC. However, E2 and TBA only stimulated proliferation rate (P<0.05) but not fusion index for CRISPR-OXT cells. Atosiban treatment resulted in lower proliferation and differentiation (P<0.05) in both wild-type BSC and CRISPR-OXT cell compared with OXT and E2 treatment groups. Together, our studies indicate that OXT plays important roles in BSC proliferation and differentiation, and it is involved in steroid hormone stimulated BSC activity. Studies to investigate specific biological mechanisms of steroid hormone stimulated OXT expression in SC are needed in the future.

No description available.

Twenty two methionine-containing di- to octa-peptides were evaluated for their ability to serve as methionine sources to support protein accretion and cell proliferation in C₂C₁₂ myogenic, MAC-T mammary epithelial and ovine myogenic satellite cells. Factors in serum that may be involved in regulating peptide utilization was investigated using MAC-T cells. Growth of MAC-T cells was studied in the presence of methionine-containing dipeptides with 6% desalted adult animal serum from chickens, horses, humans, pigs or rabbits. Serumal peptidase activities on the twenty two methionine-containing peptides were examined in cell-free, methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% fetal bovine serum. The cell cultures were incubated for 72 h at 37°C in a humidified environment of 90% air : 10% CO₂ for C₂C₁₂ and ovine satellite cells or 95% air : 5% CO₂ for MAC-T cells. The basal medium contained methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% desalted animal serum or one of the following serumal factors: .4% bovine serum lipids, 1% chemically defined lipid concentrate, bovine insulin (1 ug/mL), or 3% low protein serum replacement (LPSR-1). Treatment media tested included basal medium or basal media supplemented with L-methionine or one of the methionine-containing peptides. Cell cultures incubated with the basal media for 72 h were characterized by decreased cell number and decreased protein content compared with initial cultures. All the methionine-containing peptides (with the exception of glycylmethionine and prolylmethionine for C₂C₁₂ cells), regardles of chain length, were able to support protein accretion with responses ranging from 29 to 123% of that of free L-methionine. The DNA contents of ovine satellite cell cultures indicated that cell proliferation occurred in the presence of all the methionine-containing peptides with responses ranging from 45 to 144% of the L-methionine response. Bovine insulin and lipids were not effective in promoting peptide utilization by MAC-T cells. However, the LPSR-1 facilitated the utilization of methionine-containing peptides in C₂C₁₂ and MAC-T cells. In the cell-free, methionine-free Dulbecco’s modified Eagle’s medium, peptidases could release all the methionine residues from the tetra- to octapeptides during 24 h of incubation and 42 to 70% of the methionine residues from the di- and tripeptides tested. The results demonstrated that cultured animal cells possess the ability to utilize methionine-containing peptides as methionine sources for protein accretion and cell proliferation, but serumal peptidases are at least partially responsible for the observed responses. / Ph. D.

The overall objective of this dissertation project was to identify factors and mechanisms that control bovine myoblast proliferation, differentiation, and fusion. Three studies were conducted during this project. The objective of the first study was to determine the effect of oxytocin (OXT) on myoblast proliferation, differentiation and fusion. Treating primary bovine myoblasts in culture with 10 nM and 100 nM OXT for 24 h increased their proliferation rate by 7% (P < 0.05) and 10% (P < 0.05), respectively. Treating bovine myoblasts with either concentration of OXT for 48 h had no effect on their differentiation and fusion, as indicated by no changes in mRNA expression of selected myoblast differentiation markers and fusion index. The objective of the second study was to determine the effects of arachidonic acid (AA) and its major metabolites prostaglandin E2 (PGE2), PGF2a, and PGI2 on myoblast proliferation, differentiation and fusion. Treating myoblasts with 10 μM AA, 1 μM PGE2, 1 μM PGF2α, and 1 μM PGI2 for 24 h each increased the number of proliferating cells by 13%, 24%, 16%, and 16%, respectively, compared to the control (P < 0.05). At the same concentrations, AA, PGE2, and PGF2a stimulated myoblast differentiation and PGE2 improved myoblast fusion (P < 0.05). Treating myoblasts with AA and the cyclooxygenase (COX)-1 and COX-2 inhibitor indomethacin or the COX-2-specific inhibitor NS-398 reversed the stimulatory effect of AA on myoblast proliferation (P < 0.05). The objective of the third study was to determine the role of the proteasome in bovine myoblast differentiation and fusion. It was found that the proteasome activity increased (P < 0.05) during myoblast differentiation and fusion. Adding 5 μM lactacystin, a specific inhibitor of the proteasome, to the differentiation medium nearly completely blocked myoblast differentiation and fusion. Inhibitor of DNA-binding 1 (ID1) is known to inhibit myoblast differentiation and to be degraded by the proteasome in some cells. Both ID1 protein and mRNA expression were found to decrease during myoblast differentiation and fusion, and the decrease in ID1 protein but not ID1 mRNA was reversed (P < 0.05) by treating the cells with lactacystin. In summary, this project reveals that OXT and AA are stimulators of bovine myoblast proliferation and that AA is a stimulator of bovine myoblast differentiation. This project also indicates that the proteasome plays a positive role in bovine myoblast differentiation and fusion, and that it does so perhaps by reducing the accumulation of the ID1 protein. / Ph. D. / Myoblast proliferation, differentiation, and fusion are key steps in skeletal muscle formation and growth. Three studies were conducted to identify factors and mechanisms that control these steps in cattle, agriculturally important animals. The objective of the first study was to determine the effect of oxytocin on proliferation, differentiation and fusion of myoblasts isolated from adult cattle. Treating bovine myoblasts in culture with oxytocin increased their proliferation rate but had no effect on their differentiation and fusion. The objective of the second study was to determine the effects of arachidonic acid and its major metabolites prostaglandin E2 (PGE₂), PGF₂_a, and PGI₂ on proliferation, differentiation and fusion of bovine myoblasts. All of these chemicals were found to stimulate myoblast proliferation; AA, PGE2, and PGF2a to stimulate myoblast differentiation; and PGE₂ to improve myoblast fusion. The objective of the third study was to determine the role of the proteasome in bovine myoblast differentiation and fusion. The proteasome activity was found to increase during myoblast differentiation and fusion. Furthermore, the proteasome activity was found to promote myoblast differentiation and fusion. These results imply that skeletal muscle development and growth in cattle could be potentially improved by administrating cattle with oxytocin or prostaglandin or by altering the activity of the proteasome in cattle skeletal muscle.

No / Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27(Kip1) and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27(Kip1) knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27(Kip1) expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27(Kip1) expression, and positively regulates p27(Kip1) promoter activity, thus further supporting a role of p27(Kip1) in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.