The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear.

Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell.

The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects.

The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane.

In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

Aims/hypothesis: Several studies have suggested that large fat cells are less responsive to insulin than small fat cells. However, in these studies, large fat cells from obese individuals were compared with smaller fat cells from leaner participants, in effect making it impossible to draw conclusions about whether there is a causal relationship between fat cell size and insulin sensitivity. We hypothesised that small fat cells might be more insulin-responsive than large adipocytes when obtained from the same individual.

Materials and methods: We developed a method of sorting isolated primary human fat cells by using nylon filters of two different pore sizes. The cells were stained to visualise DNA, which allowed discrimination from artefacts such as lipid droplets. The sorted cells were left to recover overnight, since we had previously demonstrated that this is necessary for correct assessment of insulin response.

Results: We found similar amounts of the insulin receptor (IR), IRS-1 and GLUT4 when we compared small and large adipocytes from the same volunteer by immunoblotting experiments using the same total cell volume from both cell populations. Activation of IR, IRS-1 and Akt1 (also known as protein kinase B) by insulin was similar in the two cell populations. However, immunofluorescence confocal microscopy of plasma membrane sheets did not reveal any increase in the amount of GLUT4 in the plasma membrane following insulin stimulation in the large fat cells, whereas we saw a twofold increase in the amount of GLUT4 in the small fat cells.

Conclusions/interpretation: Our results support a causal relationship between the accumulation of large fat cells in obese individuals and reduced insulin responsiveness.

OBJECTIVE: We studied the activity and regulation of the peroxisome proliferator-activated receptor-gamma response element (PPRE) in primary human adipocytes.

METHODS: We transfected primary human adipocytes with a plasmid-encoding firefly luciferase cDNA under control of a PPRE from the acyl-coenzyme A oxidase gene by using our newly developed electroporation-based method. Several fatty acids were added to the fat cells to study potential activation of peroxisome proliferator-activated receptor-gamma.

RESULTS: Cells responded maximally to 5 microM of rosiglitazone at a 5.1 +/- 1.4-fold over basal increase in luciferase activity. There was a positive correlation between body mass index and the response to 5 microM of rosiglitazone (r = 0.36, P = 0.03). Patients with type 2 diabetes had similar basal PPRE activity but responded more strongly to 5 microM of rosiglitazone than did non-diabetic subjects (10.2 +/- 5-fold and 5.4 +/- 1-fold over basal increase, respectively, P < 0.0001). Among saturated fatty acids, lauric acid was without effect, but 10 microM of palmitic or stearic acid increased PPRE activity 20% to 35% above basal levels. Monounsaturated palmitoleic acid at 1 microM induced a PPRE transcriptional activity that corresponded to half the therapeutic levels of rosiglitazone.

CONCLUSION: Adipocytes from obese subjects and patients with type 2 diabetes responded particularly strongly to the effect of rosiglitazone on PPRE. Because fatty acids in the diet can affect the transcriptional activity of peroxisome proliferator-activated receptor-gamma over decades, the stimulation induced by stearic and palmitoleic acids can affect insulin sensitivity and, hence, cardiovascular morbidity and mortality in humans.

Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated signalling. Here we report that in primary human subcutaneous adipocytes the insulin receptor was localized to caveolae by electron microscopy/immunogold detection and by isolating caveolae from plasma membranes. Part of insulin receptor substrate 1 (IRS1), the immediate downstream signal mediator, was colocalized with the insulin receptor in the plasma membrane and caveolae, as demonstrated by immunofluorescence microscopy, immunogold electron microscopy, and immunogold electron microscopy of transfected recombinant HA-IRS1. In contrast, rat epididymal adipocytes lacked IRS1 at the plasma membrane. Depletion of cholesterol from the cells using β-cyclodextrin blocked insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulin stimulation of protein kinase B and Map-kinases extracellular signal-related kinase (ERK)1/2 phosphorylation. Insulin-stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1. In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes. Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines.

Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% ± 5 (mean ± S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.

Caveolae are plasma membrane invaginations with several functions, one of which appears to be to organize receptor mediated sigoaling. Here we show that in human adipocytes the iosulin receptor is localized in caveolae: by electron microscopy and immunogold detection and by isolating caveolae from plasma membranes. We similarly demonstrate that significant part of the immediate downstream signal mediator IRS1 is localized at the plasma membrane and caveolae. A detailed image shows the caveola as a bulb, protroding into the cell interior, with a neck attaching it to the plasma membrane. The caveolar structural protein caveolin is localized in the neck aod not in the bulb of the caveola. The receptor is active in caveolae since insulin stimulation caused tyrosine specific phosphorylation of the receptor recovered in isolated caveolae. Caveolae contain a major part of the free cholesterol in the plasma membrane and cholesterol is a stroctural component of caveolae. Depletion of cholesterol from the cells using B-cyclodextrio blocks insulin stimulation of glucose uptake, insulin inhibition of perilipin phosphorylation in response to isoproterenol, and insulio stimulation of protein kinase B and Map-kinases ERK1/2 phosphorylation- in effect making the human adipocytes insulin resistant. The insulin-stimulated phosphorylation of the insulin receptor and IRS1 are, however, not affected, indicating that caveolae integrity is required downstream of IRS1, consistent with its colocalization with the insulin receptor io caveolae in human adipocytes.