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Mid-lactation Holstein cows (n = 4) were used to evaluate the effects of complete feed deprivation on circulating insulin-like growth factors (IGF) and their binding proteins (IGFBP). Comparisons were made between cows in fed and unfed conditions and included response to a single injection of bovine somatotropin (bST). Two days of feed deprivation decreased milk yield 66%. Concentrations of bST nearly doubled during feed deprivation, whereas glucose and insulin steadily decreased. Circulating concentrations of IGF-I decreased nearly 50%, IGF-II decreased 28% and IGFBP-2 increased 49% during the 2 d of feed deprivation. In contrast, IGFBP-3 was not affected. Eighteen to 24 h after the bST challenge, circulating concentrations of IGF-I rose 100% during the fed period, but there was no response during the unfed period. Blood concentrations of IGF-II decreased 17% after bST challenge in the fed period but were unchanged in the unreal period. More moderate undernutrition (80% of requirements) did not affect circulating IGFBP-2 but did alter the response of IGFBP-2 to bST. A conceptual model was developed that illustrates that nutritional regulation of the ST/IGF system represents a key component signaling the appropriate use of nutrients. Overall, results demonstrate that nutritional modulation of the ST/IGF system plays a key role in animal performance and well-being over a wide range of physiological situations. INDEXING KEY WORDS: nutrition; lactation; bovine; insulin-like growth factor; somatotropin

IGF-binding protein (IGFBP)-3 is a multifunctional protein that can exert IGF-independent effects on apoptosis. Anisomycin (ANS) is a potent inducer of IGFBP-3 production in bovine mammary epithelial cells (MECs), and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. IGFBP-3 is present in the nucleus and the conditioned media in response to ANS. The goal of this study was to determine whether ribotoxic stress induced by ANS or a second ribotoxin, deoxynivalenol (DON), specifically regulates transport of IGFBP-3 to the nucleus and to determine the pathway by which it traffics. In ribotoxin-treated cells, both endogenous IGFBP-3 and transfected IGFBP-3 translocated to the nucleus. Inhibition of the nuclear transport protein importin- β with importazole reduced ribotoxin-induced nuclear IGFBP-3. Immunoprecipitation studies showed that ANS induced the association of IGFBP-3 and importin- β, indicating that ribotoxins specifically induce nuclear translocation via an importin- β ‒dependent mechanism. To determine whether secretion of IGFBP-3 is required for nuclear localization, cells were treated with Pitstop 2 or brefeldin A to inhibit clathrin-mediated endocytosis or overall protein secretion, respectively. Neither inhibitor affected nuclear localization of IGFBP-3. Although the IGFBP-3 present in both the nucleus and conditioned media was glycosylated, secreted IGFBP-3 exhibited a higher molecular weight. Deglycosylation experiments with endoglycosidase Hf and PNGase indicated that secreted IGFBP-3 completed transit through the Golgi apparatus, whereas intracellular IGFBP-3 exited from the endoplasmic reticulum before transit through the Golgi. In summary, ANS and DON specifically induced nuclear localization of nonsecreted IGFBP-3 via an importin- β ‒mediated event, which may play a role in their ability to induce apoptosis in MECs. [ABSTRACT FROM AUTHOR]

Mammary epithelial cell (MEC) number is an important determinant of milk production in lactating dairy cows. IGF-I increases IGF binding protein-3 (IGFBP-3) production in these cells, which plays a role in its ability to enhance proliferation. In the present study, we show that the apoptotic factor anisomycin (ANS) also increases IGFBP-3 mRNA and protein in a dose- and concentration-dependent manner that mirrors activation of caspase-3 and -7, with significant increases in both IGFBP-3 protein and caspase activation observed by 3 h. Knock-down of IGFBP-3 with small interfering (si) RNA attenuated the ability of ANS to induce apoptosis, while knock-down of IGFBP-2, the other major IGFBP made by bovine MEC, had no effect. Reducing IGFBP-3 also decreased the ability of ANS to induce mitochondrial cytochrome c release, indicating its involvement in the intrinsic apoptotic pathway. In contrast, transfection with IGFBP-3 in the absence of ANS failed to induce apoptosis. Since both the mitogen IGF-I and the apoptotic inducer ANS increase IGFBP-3 production in MEC, we proposed that cellular localization might determine IGFBP-3 action. While both IGF-I and ANS stimulated the release of IGFBP-3 into conditioned media, only ANS induced nuclear localization of IGFBP-3. A pan-caspase inhibitor had no effect on ANS-induced nuclear localization of IGFBP-3, indicating that nuclear entry of IGFBP-3 precedes caspase activation. Treatment with IGF-I had no effect on ANS-induced nuclear localization, but did block ANS-induced apoptosis. In summary, our data indicate that IGFBP-3 plays a role in stress-induced apoptosis that may require nuclear localization in non-transformed MEC. J. Cell. Physiol. 228: 734-742, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Ricin is a highly toxic type II ribosome-inactivating protein that has potential as a biochemical weapon and as the toxic component of immunotoxins. The unfolded protein response (UPR) is a survival response that helps cells to recover from endoplasmic reticulum (ER) stress. Failure to recover from ER stress leads to apoptosis. In yeast, ricin-A-chain (RTA), the enzymatic component of ricin, inhibits UPR. Our goals were to determine if RTA inhibits UPR in two epithelial cell lines and if this affects RTA cytotoxicity. RTA alone did not induce UPR. However, RTA inhibited both phosphorylation of inositol-requiring enzyme 1 (IRE1) and splicing of X-box binding protein1 mRNA by the UPR-inducing agent tunicamycin (Tm). The ability of dithiothreitol (DTT) to activate eukaryotic translation initiation factor 2 alpha (eIF2α), a component of the PERK pathway, was also inhibited by RTA. Treatment with RTA in combination with Tm or DTT inhibited protein synthesis more than either agent did alone in one cell line, while caspase cleavage was enhanced by the treatment combination in both cell lines. These data indicate that RTA is more cytotoxic when UPR is inhibited. This ability to inhibit UPR may enhance the potential of RTA as a therapeutic immunotoxin in solid tumors. [ABSTRACT FROM AUTHOR]

IGF binding protein (IGFBP)-3 is an important regulator of mammary epithelial cell (MEC) growth and can enhance the ability of both IGF-I and epidermal growth factor ligands such as TGFalpha to stimulate MEC proliferation. Here we investigate the role of the phosphatidylinositol-3 kinase (PI3K) and MAPK pathways in the regulation of IGFBP-3 expression by IGF-I and TGFalpha in bovine MECs. Both growth factors stimulated DNA synthesis, although IGF-I was the stronger mitogen. IGF-I and TGFalpha also stimulated IGFBP-3 mRNA and protein levels. TGFalpha stimulated rapid, transient activation of Akt that was maximal at 5 min and diminished by 15 min. In contrast, IGF-I-induced Akt activation was maximal between 15 and 90 min and was sustained for 6 h. Although ERK 1/2 was maximally stimulated by TGFalpha between 5 and 15 min, IGF-I did not stimulate discernible activation of ERK 1/2. In addition, TGFalpha but not IGF-I induced rapid phosphorylation of Shc, whereas only IGF-I activated insulin receptor substrate-1. Pretreatment with the PI3K inhibitor LY294002 or knockdown of p85 with small interfering RNA inhibited IGF-I or TGFalpha-stimulated IGFBP-3 expression. Similarly, MAPK kinase-1 inhibitors PD98059 and U0126 each abolished TGFalpha-stimulated increases in IGFBP-3 mRNA levels. In contrast to TGFalpha, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of MAPK kinase-1 inhibitors, indicating that IGF-I may activate alternative substrates of the PI3K pathway that are involved in IGFBP-3 regulation. In conclusion, stimulation of IGFBP-3 mRNA levels by mitogens is regulated through both the PI3K and MAPK pathways in bovine MECs.