Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism.

Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 µg/ml of doxycycline (dox) induced UCP-2 fourfold (424 ± 113%, mean±SEM) and 0.1 µg/ml twofold (178 ± 29%, n=3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 ± 11%) as well as D-[U-(14)C]-glucose oxidation (+5 ± 9% at 11 mM glucose). Oxidation of [1-(14)C]-oleate was increased from 4088 to 5797 fmol/µg prot/2h at 3.3mM glucose, p<0.03. Oxidation of L-[(14)C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p<0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p<0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the positive findings. A fourfold induction of UCP-2 was required to exert minor metabolic effects. These findings question an impact of moderately elevated UCP-2 levels in beta cells as seen in diabetes.

Facilitation of fatty acid uptake by CD36 in insulin-producing cells reduces fatty-acid-induced insulin secretion and glucose regulation of fatty acid oxidation.

Facilitation of fatty acid uptake in beta cells could potentially affect beta cell metabolism and secretory function; however such effects have not been clearly documented. CD36 facilitates uptake of fatty acids (FA) in muscle and adipose tissue and is likely to exert a similar effect in beta cells. We investigated the impact of over-expressing CD36 on fatty acid uptake and beta cell function by a Tet-on system in INS-1 cells. Doxycycline dose-dependently increased the CD36 protein with localization mainly in the cell membrane. Over-expression increased both specific uptake and efflux of oleate whereas intracellular glycerides were only marginally increased and incorporation of 14C-oleate or -palmitate into di- or triglycerides not affected. The normal potentiation of glucose-induced insulin secretion by acute addition of FA (50-100 micromol/l oleate and palmitate) was lost and the normal inhibitory effect of high glucose both on oleate oxidation and on the activity of carnitine palmitoyltransferase I was reduced. Over-expression did not induce apoptosis. We conclude that induction of the CD36 transporter increases uptake of FA, the consequences of which are blunting of the functional interplay between glucose and FA on insulin secretion and oxidative metabolism.

Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide.

RP105 is a B-cell surface molecule that has been recently assigned as CD180. RP105 ligation with an antibody induces B-cell activation in humans and mice, leading to proliferation and up-regulation of a costimulatory molecule, B7.2/CD86. RP105 is associated with an extracellular molecule, MD-1. RP105/MD-1 has structural similarity to Toll-like receptor 4 (TLR4)/MD-2. TLR4 signals a membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS). MD-2 is indispensable for TLR4-dependent LPS responses because cells expressing TLR4/MD-2, but not TLR4 alone, respond to LPS. RP105 also has a role in LPS responses because B cells lacking RP105 show hyporesponsiveness to LPS. Little is known, however, regarding whether MD-1 is important for RP105-dependent LPS responses, as MD-2 is for TLR4. To address the issue, we developed mice lacking MD-1 and generated monoclonal antibodies (mAbs) to the protein. MD-1-null mice showed impairment in LPS-induced B-cell proliferation, antibody production, and B7.2/CD86 up-regulation. These phenotypes are similar to those of RP105-null mice. The similarity was attributed to the absence of cell surface RP105 on MD-1-null B cells. MD-1 is indispensable for cell surface expression of RP105. A role for MD-1 in LPS responses was further studied with anti-mouse MD-1 mAbs. In contrast to highly mitogenic anti-RP105 mAbs, the mAbs to MD-1 were not mitogenic but antagonistic on LPS-induced B-cell proliferation and on B7.2 up-regulation. Collectively, MD-1 is important for RP105 with respect to B-cell surface expression and LPS recognition and signaling.