Figure 3 The subcellular distribution of LPL activity in 3T3-L1 adipocytes. Adipocytes were made insulin resistant (desensitized) by treatment with 100 nm insulin for 18 h. The treated cells were then exhaustively washed to remove the desensitizing dose of insulin. Basal, naive adipocytes (left panel) or desensitized adipocytes (right panel) were treated in the absence (solid bar) or presence (hatched bar) of 100 nm insulin for 60 min. Cell culture medium was collected from the cells and assayed for LPL activity (Medium). Culture medium containing 100 μg/ml heparin was immediately placed upon the cells. After 20 min, the heparin-containing medium was collected and assayed for LPL activity (Hep). The cell monolayers were then washed with PBS, and the residual LPL activity associated with the cells was determined, as described in Materials and Methods (Cell). Statistically significant differences in values obtained from insulin-free and insulin-treated cells are denoted by an asterisk. From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society

Figure 2 The heparin-induced release of LPL from 3T3-L1 adipocytes Figure 2 The heparin-induced release of LPL from 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated with heparin, and the LPL activity released into the cell culture medium was quantitated. A, The time course of heparin-induced release of LPL. Adipocytes were incubated in the absence (○) or presence (•) of 100μ g/ml heparin for the indicated periods of time. The cell culture medium bathing the cells was then collected and assayed for LPL activity. B, Heparin concentration-response profile for the release of LPL from adipocytes. The indicated concentrations of heparin were incubated with the adipocytes for 20 min. The cell culture medium was then collected and assayed for LPL activity. From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society

Figure 1 The insulin-induced release of LPL from 3T3-L1 adipocytes Figure 1 The insulin-induced release of LPL from 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were treated with insulin, and LPL activity released into the cell culture medium was quantitated. A, Time course of insulin-induced release of LPL. Adipocytes were incubated in the absence (○) or presence (•) of 100 nm insulin for the indicated periods of time. The cell culture medium was then collected and assayed for LPL activity. B, Insulin concentration response for the release of LPL from the adipocytes. The adipocytes were incubated with the indicated concentration of insulin for 60 min. The cell culture medium bathing the cells was then collected and assayed for LPL activity. From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society

Figure 4 The insulin-induced release of an LPL release factor from endothelial cells. Confluent HAE cells were incubated with (•) or without (○) 100 nm insulin for the indicated periods of time. The endothelial cell-conditioned cell culture medium was then immediately added to 3T3-L1 adipocytes and incubated for 60 min. The culture medium was then removed from the adipocytes and assayed for LPL activity. From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society

Figure 5 The insulin-induced release of an LPL release factor from macrovessel, bovine aorta endothelial cells. Early passage BAE cells were cultured under the conditions described for HAE cells. Confluent cultures were incubated with (•) or without (○) 100 nm insulin for the indicated periods of time. The endothelial cell-conditioned cell culture medium was immediately added to 3T3-L1 adipocytes and incubated for 60 min. The culture medium was then harvested from the adipocytes and assayed for LPL activity. From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society

Figure 6 The insulin-induced release of the LPL release factor from microvessel endothelial cells. A, The insulin concentration-response profile of the production of the LPL release factor by endothelial cells. HAE cells were incubated with the indicated concentration of insulin for 20 min. The cell culture medium was removed from the endothelial cells, added to 3T3-L1 adipocytes, and allowed to incubate for 60 min. The cell culture medium was then removed from the adipocytes and assayed for LPL activity. The open circle denotes LPL activity released from the adipocytes by insulin alone, in the absence of the endothelial cell conditioned medium. B, Time course of the release of LPL activity from adipocytes by the endothelial cell-derived LPL release factor. Endothelial cells were incubated with 10 nm insulin for 20 min. The conditioned medium was then removed from the endothelial cells and placed on the adipocytes for the indicated periods of time. The medium was removed from the adipocytes and assayed for LPL activity. The open circle denotes the level of LPL released from the adipocytes by 10 nm insulin alone in the absence of endothelial cell-derived factors. From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society

Figure 7 The effect of repeated insulin challenge on the ability of the endothelial cells to produce the LPL release factor. Basal, naive endothelial cells were incubated with 10 nm insulin for the indicated periods of time, and culture medium bathing the cells was collected and added to adipocytes for 60 min. Medium was then collected from the adipocytes and assayed for LPL activity (•). The same endothelial cells were washed to remove any residual insulin, and then incubated in basal medium for 2.5 h. The endothelial cells were rechallenged with insulin, the endothelial cell-conditioned medium was incubated with adipocytes, and the resultant medium was assayed for LPL activity (○). A second set of endothelial cells was incubated with 100 nm insulin for 18 h. These desensitized endothelial cells were washed to remove insulin and were then incubated with 10 nm insulin for the indicated periods of time. The conditioned medium was removed from the desensitized endothelial cells and added to adipocytes for 60 min, and then the medium was removed from the adipocytes and assayed for LPL activity (▴). A parallel set of endothelial cells was incubated in the absence of insulin for the indicated periods of time, and after incubation of this conditioned medium with the adipocytes for 60 min, the resultant medium was assayed for LPL activity (▵). From: The Release of Lipoprotein Lipase from 3T3-L1 Adipocytes Is Regulated by Microvessel Endothelial Cells in an Insulin-Dependent Manner Endocrinology. 2000;141(2):693-701. doi:10.1210/endo.141.2.7315 Endocrinology | Copyright © 2000 by The Endocrine Society