Hyperglycemia and advanced glycosylation end products suppress adipocyte apoE expression: implications for adipocyte triglyceride metabolism

Abstract

Endogenous adipocyte apolipoprotein E (apoE) plays an important role in adipocyte lipoprotein metabolism and lipid flux. A potential role for hyperglycemia in regulating adipocyte apoE expression and triglyceride metabolism was examined. Exposure of adipocytes to high glucose or advanced glycosylation end product-BSA significantly suppressed apoE mRNA and protein levels. This suppression was significantly attenuated by antioxidants or inhibitors of the NF-κB transcription pathway. Hyperglycemia in vivo led to adipose tissue oxidant stress and significant reduction in adipose tissue and adipocyte apoE mRNA level. Incubation with antioxidant in organ culture completely reversed this suppression. Hyperglycemia also reduced adipocyte triglyceride synthesis, and this could be completely reversed by adenoviral-mediated increases in apoE. To more specifically evaluate an in vivo role for adipocyte apoE expression on organismal triglyceride distribution in vivo, WT or apoE knockout (EKO) adipose tissue was transplanted in EKO recipient mice. After 12 wk, WT adipocytes transplanted in EKO mice accumulated more triglyceride compared with transplanted EKO adipocytes. In addition, EKO recipients of WT adipose tissue had reduced hepatic triglyceride content compared with EKO recipients transplanted with EKO adipose tissue. Our results demonstrate that hyperglycemia and advanced glycosylation end products suppress the expression of adipocyte apoE in vitro and in vivo and thereby reduce adipocyte triglyceride synthesis. In vivo results using adipose tissue transplantation suggest that reduction of adipocyte apoE, and subsequent reduction of adipocyte triglyceride accumulation, could influence lipid accumulation in nonadipose tissue.

Fig. 1.

High glucose decreases adipocyte apolipoprotein E (apoE) mRNA and protein expression. 3T3-L1 cells were differentiated using the hormone cocktail described in materials and methods. After being maintained for 6 days in 10% FBS/DMEM with 5 mM glucose, cells were incubated for an additional 2 days in fresh medium containing 5 or 25 mM glucose. A: apoE mRNA level was measured using quantitative RT-PCR. B: representative immunoblot and quantitation showing apoE expression in adipocytes incubated in high or low glucose. β-Actin was used to correct for protein loading. Results shown are means ± SD of triplicate samples. *P < 0.01 for the difference between 5 and 25 mM glucose.

Fig. 2.

Antioxidants and NF-κB pathway inhibitors prevent suppression of adipocyte apoE expression by high glucose. Mature 3T3-L1 adipocytes were incubated in medium containing 5 or 25 mM glucose over 2 days as indicated. N-acetyl-l-cysteine (NAC, 20 mM; A and B) or 10 nmol 6-amino-4-(4a-phenoxyphenylethylamino)quinazoline (QNZ, C and D) were included as indicated. apoE mRNA levels are shown in A and C. Representative immunoblots with quantitation are presented in B and D. Results shown are means ± SD of triplicate samples. *P < 0.01 for differences between 5 and 25 mM glucose. #P < 0.01 for the effect of NAC or QNZ.

Fig. 3.

Advanced glycosylation end products (AGE) suppress adipocyte apoE expression. Mature 3T3-L1 adipocytes were incubated in 5 mM DMEM containing 0.1% FBS with 0.3 mg/ml BSA or AGE-modified BSA for 18 h. A: apoE mRNA level was measured as described in materials and methods. B: representative immunoblot and quantitation. Results shown are means ± SD of triplicate samples. *P < 0.01 for the difference between control and AGE-modified BSA.

Fig. 4.

Pathways mediating regulation of adipocyte apoE gene expression by AGE. Control or AGE-BSA (0.3 mg/ml) was added to mature 3T3-L1 cells in the presence or absence of NAC (A) or QNZ (B) for 18 h before harvest of cells for measurement of apoE mRNA. C: AGE-modified BSA was added to mature 3T3-L1 cells for the times indicated before measurement of apoE mRNA. D: cells were incubated with control or AGE-BSA (0.3 mg/ml) for 6 h with or without inclusion of a neutralizing antibody for the receptor for advanced glycosylation end product (RAGE). Values shown are means ± SD of triplicate samples. *P < 0.05 for control compared with AGE-BSA. #P < 0.01 for the effect of NAC, QNZ, or the RAGE neutralizing antibody.

Fig. 5.

Hyperglycemia in vivo suppresses adipose tissue and adipocyte apoE expression. Freshly isolated adipose tissue (A–C) or adipocytes (B) from control or streptozotocin (STZ)-treated diabetic mice was used for measurement of reactive oxygen species (ROS; A) or of apoE and CD68 mRNA levels (B). C: freshly collected adipose tissue from control or STZ diabetic mice was maintained in organ culture in the presence or absence of 20 mM NAC for 6 h. At that time, the tissue was washed with PBS, and apoE mRNA levels were measured. Results shown are means ± SD of adipose tissue collected from five separate mice. *P < 0.01 for the difference between control and diabetic mice. #P < 0.01 for the effect of NAC.

Fig. 6.

Impact of hyperglycemia-induced apoE suppression on adipocyte triglyceride (Tg) synthesis. A: 3T3-L1 adipocytes were incubated in high or low glucose as described in the legend to Fig. 1. At the end of this incubation, triglyceride synthesis was measured during a 2-h incubation in [¹⁴C]oleic acid as described in materials and methods. B: 3T3-L1 cells were incubated in low glucose or high glucose and transduced with an adenovirus expressing LacZ or apoE as indicated. Western blot for cellular apoE was performed as described in materials and methods. C: 3T3-L1 cells were incubated in high or low glucose with the indicated adenovirus exactly as done in B and then used for measurement of triglyceride synthesis. *P < 0.05 and **P < 0.01. OA, oleic acid.

Fig. 7.

Adipose tissue and liver triglyceride parameters in apoE knockout (EKO) mice transplanted with wild-type (WT) or EKO adipose tissue. Intra-abdominal adipose tissue harvested from WT or EKO mice was transplanted in EKO recipients (6 mice in each transplantation group). For each transplantation pair indicated, the first symbol indicates the genotype of the adipose tissue donor. After 12 wk, mice were euthanized for harvest of transplanted adipose tissue, liver, and soleus muscle. A: hematoxylin- and eosin-stained sections of WT and EKO transplanted adipose tissue; original magnification ×200. B: mature adipocytes were isolated from transplanted adipose tissue and sized as described in materials and methods. Size distribution for mature WT and EKO adipocytes isolated from transplanted adipose tissue is shown. C, left: mean adipocyte volume for transplanted EKO vs. WT adipocytes. C, right: total adipocyte lipid per cell in transplanted EKO vs. WT adipocytes. D: freshly isolated mature adipocytes from transplanted EKO or WT adipose tissue were used for measurement of triglyceride synthesis as described in materials and methods. E: triglyceride mass was measured in liver and muscle harvested from transplantation recipients. *P < 0.05 and **P < 0.01.