Cristina M. Rondinone

Immunocytochemistry was used to determine the intracellular location of perilipins in adipocytes and the occurrence of these proteins in tissues involved in triacylglycerol metabolism. Confocal microscopy and 3-dimensional analysis of 3T3-L1 adipocytes showed that perilipin immunofluorescence, present on the surfaces of all sized lipid droplets, appeared unevenly dispersed on the surfaces of many large lipid droplets. Electron microscopy revealed that immunogold staining for perilipin was located directly on the surface layer apposed to and surrounding the core triacylglycerol of intracellular lipid droplets of adipocytes in culture or from white and brown adipose tissue. Freeze-fracture electron microscopy indicated that the hydrophobic face of this surface monolayer contained particles identical in size and distribution to intramembranous particles (IMPs), which are unique structural features of the hydrophobic faces of bilayered membranes. Also, freeze-fracture replicas revealed areas of continuity between the surface layer of lipid droplets and the membrane leaflets of endoplasmic reticulum, suggesting that the droplet monolayer surface is an area of endoplasmic reticulum membrane leaflet modified by its unique content of perilipin. Microperoxisomes, identified by immunostaining for catalase, were found closely associated with lipid droplets, but external to and not in contact with the lipid droplet surface layer. Vimentin, identified by immunofluorescence, was present around the periphery of most lipid droplets in 3T3-L1 cells during early stages of adipocyte development but, in contrast to perilipins, vimentin was not around the periphery of many large lipid droplets in mature cells. Although perilipin was at the surface of lipid droplets in adipocytes of lactating mammary gland, none was found to be associated with the milk lipid droplets in alveolar epithelial cells, nor was the protein found on the surfaces of lipid droplets in hepatocytes. Studies in mammary gland show that perilipin immunostaining will be a valuable tool for the identification of tissue adipocytes severely depleted of their triacylglycerol stores and thus without their characteristic spherical shape. Perilipin's singular location on the surface monolayer of intracellular lipid droplets supports an intimate role for the protein in the triacylglycerol metabolic functions of adipocytes.

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity.