PI4KIIIα is targeted to the plasma membrane via an evolutionarily conserved complex comprised of EFR3 and TTC7 to control PtdIns4P synthesis and the selective enrichment of PtdIns(4,5)P2 in this membrane.

Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents.

Lipids and other membrane constituents recycle between the plasma membrane and intracellular endocytic compartments. In CHO cells, approximately half of the internalized C 6 -NBD-SM, a fluorescent lipid analogue widely used as a membrane maker, recycles via the endocytic recycling compartment with a t1 ⁄2 of ϳ12 min (Mayor, S., Presley, J. F., and Maxfield, F. R. (1993) J. Cell Biol. 121, 1257-1269). Surprisingly, the rest returns to the plasma membrane very quickly. A detailed kinetic study presented in this paper indicates that after a brief internalization pulse, 42-62% of the internalized C 6 -NBD-SM returns to the plasma membrane with a t1 ⁄2 of 1-2 min. Similar results are obtained using HEp2 and nonpolarized Madin-Darby canine kidney cells. Using FM dyes of different hydrophobicity, we show that rapid recycling involves passage through an endocytic organelle that was subsequently identified as the sorting endosome by co-localization with internalized transferrin and low density lipoprotein. These results imply that the membrane internalization rate is much higher than previously estimated, with a t1 ⁄2 as short as 5-10 min. Rapid internalization and recycling would facilitate processes such as nutrient uptake and cholesterol efflux.Endocytic recycling is essential for regulation of surface expression of proteins and for the uptake of nutrients. After internalization, endocytosed molecules are delivered rapidly to sorting endosomes (1, 2), which consist of vesicles with tubular extensions that are involved in transport of recycling material (e.g. transferrin receptor) out of the sorting endosomes. A major recycling pathway involves subsequent passage through the endocytic recycling compartment (ERC), 1 which in CHO cells is a collection of tubules concentrated near the centriole, from which molecules recycle back to the plasma membrane (t1 ⁄2 ϳ9 -12 min) (3-5).Fluorescent lipid probes are very well suited for kinetic studies of endocytic recycling. Very bright signals can be obtained, and after internalization pulses, efficient desorption of certain lipid analogs from the plasma membrane allows accurate measurements of recycling with minimal interference from probe molecules left in the plasma membrane (6). After nonselective internalization, a fluorescent lipid analog, C 6 -NBDsphingomyelin (C 6 -NBD-SM), exits sorting endosomes, enters the ERC, and then returns to the plasma membrane with kinetics indistinguishable from transferrin (Tf) in CHO cells (4). After a 10-min internalization pulse with C 6 -NBD-SM, the efflux kinetics from CHO cells suggested the existence of a second, faster recycling pathway, in addition to the pathway through the ERC with a t1 ⁄2 of ϳ12 min (7). Taking full advantage of the properties of lipid analogs, we have now characterized rapid kinetics of membrane recycling in various cell types, and we found that nearly half of the internalized membrane recycles with a t1 ⁄2 of about 1.5 min. This surprisingly rapid recycling requires internalization of the lipids with a t1 ⁄2 of 5-1...

OBJECTIVE-Type 2 diabetes is often accompanied by abnormal blood lipid and lipoprotein levels, but most studies on the link between hyperlipidemia and diabetes have focused on free fatty acids (FFAs). In this study, we examined the relationship between cholesterol and insulin secretion from pancreatic ␤-cells that is independent of the effects of FFAs. RESEARCH DESIGN AND METHODS-Several methodswere used to modulate cholesterol levels in intact islets and cultured ␤-cells, including a recently developed mouse model that exhibits elevated cholesterol but normal FFA levels. Acute and metabolic alteration of cholesterol was done using pharmacological reagents.RESULTS-We found a direct link between elevated serum cholesterol and reduced insulin secretion, with normal secretion restored by cholesterol depletion. We further demonstrate that excess cholesterol inhibits secretion by downregulation of metabolism through increased neuronal nitric oxide synthase dimerization.CONCLUSIONS-This direct effect of cholesterol on ␤-cell metabolism opens a novel set of mechanisms that may contribute to ␤-cell dysfunction and the onset of diabetes in obese patients.

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Background: GLUT4 glucose transporters are trapped and sequestered intracellularly in adipocytes by TUG. Results: Insulin stimulates TUG cleavage, which separates regions of TUG that bind GLUT4 and Golgi matrix proteins. Cleavage is required for highly insulin-responsive GLUT4 translocation. Conclusion: TUG proteolysis liberates GLUT4 trapped at the Golgi matrix. Significance: Endoproteolytic cleavage is a novel biochemical mechanism for insulin action to regulate glucose uptake.

Competing interests Cornell University has filed a provisional patent application that covers the use of DUSP26 inhibitors for the treatment of type 2 diabetes. (US patent application no. 62/740744; N.G.-B. and J.C.L.). V.B. is currently an employee of Fractyl Laboratories Inc. and all analyses were conducted during employment at Massachusetts General Hospital.

We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. By using DHE loaded on methyl-␤-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface.