Introduction

The long-term complications associated with diabetes such as heart disease, kidney failure, blindness, and limb amputations are mostly the result of chronic elevations in blood glucose levels, also known as chronic hyperglycemia (1,2). The exact molecular mechanisms by which hyperglycemia causes damage to various tissues are unknown. However, it is known that chronic hyperglycemia results in acute metabolic abnormalities such as insulin resistance that leads to worsening of diabetes (2). Insulin resistance results in decreased insulin-stimulated glucose transport into skeletal muscle and adipocyte tissue (3).

Muscle and adipose tissue are insulin-responsive tissues and express the insulin-sensitive glucose transporter GLUT-4 (4). The glucose transporter GLUT-4 translocates from intracellular vesicles to the plasma membrane in response to insulin and causes increased glucose transport into muscle and fat cells (5). Because insulin resistance (decreased GLUT-4 translocation to plasma membrane in response to insulin) is one of the important factors causing NIDDM (non-insulin-dependent diabetes mellitus) (6), it is important to understand how insulin stimulates glucose uptake into insulin-responsive tissues.

3T3-L1 adipocytes are the most preferred cell line to study insulin-stimulated glucose uptake, as most of the available muscle cell lines are not insulin sensitive in terms of glucose transport. 3T3-L1 adipocytes behave like primary adipocytes in many aspects and provide an excellent model system to study

Fig. 1. 3T3-L1 preadipocyte differentiation. The fibroblast like 3T3-L1 preadi-pocytes (A) differentiate into adipocytes when incubated with insulin, dexamethasone, and 1-isobutyl-1-methylxanthine in the presence of serum. The terminally differentiated adipocytes are spherical in shape and accumulate fat droplets (B). The preadipocytes and differentiated adipocytes were infected with a GFP adenovirus and the photographs were taken using a fluorescence microscope.

Fig. 1. 3T3-L1 preadipocyte differentiation. The fibroblast like 3T3-L1 preadi-pocytes (A) differentiate into adipocytes when incubated with insulin, dexamethasone, and 1-isobutyl-1-methylxanthine in the presence of serum. The terminally differentiated adipocytes are spherical in shape and accumulate fat droplets (B). The preadipocytes and differentiated adipocytes were infected with a GFP adenovirus and the photographs were taken using a fluorescence microscope.

insulin action and signaling (7,8). This chapter describes an optimized protocol to differentiate the 3T3-L1 fibroblasts (preadipocytes) into adipocytes and the use of these terminally differentiated adipocytes to measure insulin-stimulated glucose uptake. The preadipocytes are derived from mouse embryonic tissue, exhibit a fibroblast phenotype (see Fig. 1A), and are not insulin sensitive (9). Treating the preadipocytes with a differentiation medium containing insulin, dexamethasone, and 1-isobutyl-1-methylxanthine in the presence of serum induces these cells to become terminally differentiated adipocytes. The preadipocytes then convert to a spherical shape and accumulate fat droplets (see Fig. 1B), and they progressively acquire the morphological and biochemical characteristics of a mature white adipocyte (9). Unlike preadipocytes, which express only the non-insulin-sensitive glucose transporter GLUT-1, fully differentiated 3T3-L1 adipocytes also express the insulin-responsive glucose transporter GLUT-4 (10) and glucose uptake can be activated as much as 15- to 20-fold by insulin (11). The glucose uptake assay using 3T3-L1 adipocytes is usually performed at d 10-12 after initiation of adipocyte differentiation. The adipocytes are first starved of serum, washed, and incubated with insulin to activate GLUT-4 translocation to the plasma membrane. Glucose uptake is measured using radioactive 2-deoxyglucose that cannot be metabolized by the cells. The cells are washed in ice-cold phosphate-buffered saline (PBS) and after lysis used to measure the amount of radioactive 2-deoxyglucose taken up by the cells. The amount of radioactive 2-deoxyglucose taken up by cells incubated without insulin (basal glucose uptake resulting from the GLUT-1 transporter) is subtracted from the amount of radioactivity taken up by cells incubated with insulin (insulin-stimulated glucose uptake mainly resulting from translocation of GLUT-4) giving the true insulin-stimulated glucose uptake value.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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