Macropinocytosis is decreased in diabetic mouse macrophages and is regulated by AMPK

<p>Abstract</p> <p>Background</p> <p>Macrophages (MΦs) utilize macropinocytosis to integrate immune and metabolic signals in order to initiate an effective immune response. Diabetes is characterized by metabolic abnormalities and altered immune function. Here we examine the influence of diabetes on macropinocytosis in primary mouse macrophages and in an <it>in vitro </it>diabetes model.</p> <p>Results</p> <p>The data demonstrate that peritoneal MΦs from diabetic (<it>db/db</it>) mice had reduced macropinocytosis when compared to MΦs from non-diabetic (<it>db/+</it>) mice. Additionally, MΦs cultured in hyperglycemic conditions were less adept at macropinocytosis than those cultured in low glucose. Notably, AMP-activated protein kinase (AMPK) activity was decreased in MΦs cultured in hyperglycemic conditions. Activation of AMPK with leptin or 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) increased macropinocytosis and inhibition of AMPK with compound C decreased macropinocytosis.</p> <p>Conclusion</p> <p>Taken together, these findings indicate that MΦs from diabetic mice have decreased macropinocytosis. This decrease appears dependent on reduced AMPK activity. These results demonstrate a previously unrealized role for AMPK in MΦs and suggest that increasing AMPK activity in diabetic MΦs could improve innate immunity and decrease susceptibility to infection.</p

Phagocytosis of Cholesteryl Ester Is Amplified in Diabetic Mouse Macrophages and Is Largely Mediated by CD36 and SR-A

Type 2 diabetes (T2D) is associated with accelerated atherosclerosis, which accounts for approximately 75% of all diabetes-related deaths. Here we investigate the link between diabetes and macrophage cholesteryl ester accumulation. When diabetic (db/db) mice are given cholesteryl ester intraperitoneally (IP), peritoneal macrophages (PerMΦs) recovered from these animals showed a 58% increase in intracellular cholesteryl ester accumulation over PerMΦs from heterozygote control (db/+) mice. Notably, PerMΦ fluid-phase endocytosis and large particle phagocytosis was equivalent in db/+and db/db mice. However, IP administration of CD36 and SR-A blocking antibodies led to 37% and 25% reductions in cholesteryl ester accumulation in PerMΦ. Finally, in order to determine if these scavenger receptors (SRs) were part of the mechanism responsible for the increased accumulation of cholesteryl esters observed in the diabetic mouse macrophages, receptor expression was quantified by flow cytometry. Importantly, db/db PerMΦs showed a 43% increase in CD36 expression and an 80% increase in SR-A expression. Taken together, these data indicate that direct cholesteryl ester accumulation in mouse macrophages is mediated by CD36 and SR-A, and the magnitude of accumulation is increased in db/db macrophages due to increased scavenger receptor expression

<i>Db/db</i> mice have elevated blood glucose and serum insulin levels.

<p>Fasting blood glucose (FBG), random blood glucose (RBG), fasting serum insulin (FSI), and random serum insulin (RSI). Results represent the average of n = 5 mice±SEM. *: p<0.05.</p

Phagocytosis and fluid-phase endocytosis are not increased in PerMΦs from <i>db/db</i> mice.

<p><i>A</i>, <i>Db</i>/+and <i>db/db</i> mice were IP administered 0.21 or 2.6 µm latex microspheres (beads) at 5×10⁹ or 5×10⁷ beads/ml, respectively. Following a 4 h <i>in vivo</i> incubation period, PerMΦs were collected and microsphere uptake was measured by flow cytometry. <i>B, Db</i>/+and <i>db/db</i> mice were IP administered 0.25 mg/ml FITC-dextran. Following a 4 h <i>in vivo</i> incubation period, PerMΦs were collected and dextran uptake was measured by flow cytometry. Results for <i>A</i> and <i>B</i> represent the average of three independent experiments±SEM.</p

PerMΦs from <i>db/db</i> mice have increased AcLDL and cholesteryl ester uptake.

<p><i>A, db</i>/+and <i>db/db</i> mice were administered 5 µg/mL of Dil-AcLDL by IP injection. After 4 h, PerMΦs were collected and Dil-AcLDL uptake measured by flow cytometry. <i>B</i>, db/+and db/db mice were administered 5 µg/mL of bodipy-cholesteryl ester by IP injection. After 4 h, PerMΦs were collected and cholesteryl ester uptake measured by flow cytometry. <i>C</i>, bodipy-cholesteryl ester (C.E) or carrier (Con) were administered as in A for 4 h. PerMΦs cholesteryl ester uptake was examined using fluorescent microscopy. Results are representative of three independent experiments. <i>D</i>, PerMΦs were collected from db/+and db/db mice and cultured for 30 min ex vivo. Cells were then incubated with 5 µg/mL bodipy-cholesteryl ester (C.E.) and cholesteryl ester uptake measured by flow cytometry at 4 h. Results for A, B and D represent the average of three independent experiments±SEM. *: p<0.05.</p

CD36 and SR-A mediate increased AcLDL and cholesteryl ester uptake in diabetic PerMΦs.

<p><i>A,</i> PerMΦs were collected from <i>db</i>/+mice and cultured for 30 min ex vivo. Cells were then incubated on ice for 15 m with CD36, SR-A blocking antibodies (Ab) or isotype control antibodies (Iso) as indicated. Cells were then incubated with 5 µg/ml Dil-AcLDL and uptake measured by flow cytometry at 4 h. <i>B,</i> PerMΦs were collected from <i>db/db</i> mice and cultured for 30 min ex vivo. Cells were then incubated on ice for 15 m with CD36, SR-A blocking antibodies (Ab) or isotype control antibodies (Iso) as indicated. Cells were then incubated with 5 µg/ml Dil-AcLDL and uptake measured by flow cytometry at 4 h. <i>C, Db</i>/+mice were IP administered 5 µg/ml bodipy-cholesteryl ester (C.E.) with CD36, SR-A blocking antibodies (Ab) or isotype control antibodies (Iso) as indicated. After 4 h, PerMΦs were collected and cholesteryl ester uptake measured by flow cytometry. <i>D, Db/db</i> mice were IP adminsitered 5 µg/ml bodipy-cholesteryl ester (C.E.) with CD36, SR-A blocking antibodies (Ab) or isotype control antibodies (Iso) as indicated. After 4 h, PerMΦs were collected and cholesteryl ester uptake measured by flow cytometry. <i>E</i>, PerMΦs were collected from <i>db</i>/+and <i>db/db</i> mice. CD36 and SR-A surface expression was measured by flow cytometry. Results for <i>A-E</i> represent the average of three independent experiments±SEM. *: p<0.05</p