β-cell metabolic alterations under chronic nutrient overload in rat and human islets

The aim of this study was to assess multifactorial β-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of lipid droplets in β-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in β-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 μM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.     

葛根素对2型糖尿病大鼠胰岛素抵抗及脂肪分化相关蛋白基因表达的影响

目的 观察葛根素对实验性2型糖尿病（2-DM）大鼠胰岛素抵抗及脂肪分化相关蛋白（ADRP）基因mRNA表达的影响. 方法 采用高糖高脂饮食伴尾静脉注射小剂量链脲佐菌素（STZ）复制2-DM大鼠模型. 将大鼠随机分为正常对照组、2-DM模型组和高、中、低剂量药物组（分别腹腔注射葛根素注射液160, 80, 40 mg&#8226;kg^-1&#8226;d^-1）. 6周后, 测定空腹血糖（FPG）、血脂、血清胰岛素（FINS）, 并计算胰岛素敏感指数（ISI）, 同时以逆转录 聚合本酶链反应（RT-PCR）法检测脂肪组织ADRP基因mRNA水平. 结果 与2-DM模型组比较, 高、中、低剂量药物组FPG、FINS、游离脂肪酸（FFA）、总胆固醇（TC）、三酰甘油（TG）含量显著降低, ISI明显升高（^P<0.05或^P<0.01）; 高和中剂量药物组大鼠脂肪组织ADRP基因mRNA表达显著降低. 结论 葛根素可通过下调2-DM大鼠脂肪组织ADRP基因mRNA表达, 降低胰岛素抵抗水平, 改善脂代谢紊乱.     

非酒精性脂肪性肝病发生中Perilipin和ADRP表达变化实验研究

目的：探讨高脂饮食诱导大鼠非酒精性脂肪性肝病（NAFLD）形成过程中，肝组织Perilipin（脂滴包被蛋白）和ADRP（脂肪分化相关蛋白）的表达变化及意义。方法：建立大鼠高脂饮食NAFLD模型并设正常对照组，观察时间点为建模后4w、8w和12w，观察大鼠肝组织病理变化，检测血浆游离脂肪酸及甘油三酯水平，免疫组织化学染色法检测肝组织中Perilipin和ADRP蛋白水平。结果：成功制作大鼠NAFLD实验模型，与正常组相比，NAFLD组大鼠血浆FFA水平和TG水平从第4w起显著升高（P〈0．01），NAFLD组大鼠肝组织Perilipin蛋白表达水平显著低于正常组（P〈0．01），ADRP蛋白表达水平显著高于同期正常组（P〈0．01）。Perilipin及ADRP表达与FFA及TG具有显著相关性。结论：高脂饮食可制作NAFLD大鼠模型，Perilipin和ADRP与NAFLD的形成有关。     

β-cell metabolic alterations under chronic nutrient overload in rat and human islets

The aim of this study was to assess multifactorial β-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of lipid droplets in β-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in β-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 μM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.     

High glucose,insulin and free fatty acid concentrations synergistically enhance perilipin 3 expression and lipid accumulation in macrophages

Objective

Perilipin (PLIN) 3, an intracellular lipid droplet (LD)-associated protein, is implicated in foam cell formation. Since metabolic derangements found in metabolic syndrome, such as high serum levels of glucose, insulin and free fatty acids (FFAs), are major risk factors promoting atherosclerosis, we investigated whether PLIN3 expression is affected by glucose, insulin and oleic acid (OA) using RAW264.7 cells.

Methods

Real-time PCR and Western blotting were performed to detect PLIN3 or PLIN2 expression. Oil-red O staining and Lipid Analysis were employed to measure cellular content of triacylglycerides (TAG) and cholesterol.

Results

PLIN3 mRNA was stimulated by high glucose or insulin concentrations individually, but not by OA. A combination of any two factors did not enhance PLIN3 expression any more than that evoked by glucose alone at 24 h. Interestingly, however, simultaneous addition of all three factors synergistically enhanced the PLIN3 expression. This synergistic effect was not apparent for PLIN2 mRNA expression. Inhibitors of Src family tyrosine kinase and/or phosphatidylinositol 3-kinase, both of which are activated by insulin and FFA signaling, partially suppressed PLIN3 expression induced by the combination of the three factors. While simultaneous addition of glucose, insulin and OA remarkably increased the cellular content of TAG and cholesterol, knocking-down of PLIN3 predominantly reduced TAG content.

Conclusions

These results indicate that PLIN3 expression is synergistically stimulated by high glucose, insulin and FFA concentrations, in parallel with TAG accumulation in macrophages. This finding raises new evidence of PLIN3 involvement in conversion of macrophages into foam cells     

Oncocytic adrenal cortical tumor with cytoplasmic inclusions and hyaline globules

Adrenal cortical tumors, particularly oncocytic tumors, have been reported to contain a variety of intracytoplasmic and intramitochondrial inclusions. Oncocytic cortical tumors can also morphologically mimic pheochromocytomas. We report an unusual, partially oncocytic cortical neoplasm with nesting architecture, intranuclear inclusions, and hyaline globules reminiscent of pheochromocytoma, together with numerous, small, brightly eosinophilic, periodic acid-Schiff-positive cytoplasmic inclusions and typical cytoplasmic lipid droplets. Ultrastructural study revealed oncocytes containing numerous mitochondria with intramitochondrial crystals and lipid droplets. Immunohistochemistry and immunoblots were utilized to further characterize the tumor. Immunohistochemistry demonstrated immunoreactivity of both the eosinophilic inclusions and the hyaline globules for adipose differentiation-related protein (ADRP), which is one of a group of proteins associated with storage of neutral lipids in many cell types. Immunoblots confirmed the presence of ADRP and demonstrated an imbalance between ADRP and perilipin, another neutral lipid-associated protein, in tumor tissue compared to normal adrenal cortex. The findings suggest that mitochondrial dysfunction in oncocytic cortical tumors may lead to abnormal processing of proteins related to the lipid-storing functions of the adrenal cortex, resulting in unusual cytoplasmic inclusions and extracellular globules resembling the globules in pheochromocytomas. The finding of ADRP as a constituent of inclusions in adrenal cortical tumors has not been previously reported.     

Presence of adipose differentiation-related protein in rat meibomian gland cells

Adipose differentiation-related protein (ADRP) is an intrinsic lipid storage protein found in lipid droplets of different type of cells. ADRP has been recognized to be a specific marker of lipid accumulation and a marker of differentiated adipocytes. The purpose of this study was to determine whether ADRP was present in the cells of the meibomian gland. The expression of the mRNA of ADRP was determined by RT-PCR and Northern blot analysis of the meibomian gland and other rat tissues. A newly generated polyclonal antibody against rat ADRP was used for Western blot analysis and immunohistochemical staining to determine whether ADRP was expressed in the rat meibomian gland. Meibomian gland acinar cells were isolated to determine when ADRP was expressed during cell differentiation in vitro. Northern blot analysis and Western analysis showed that ADRP was expressed in the meibomian gland. Immunoreactivity to ADRP was observed in the lobules of acinar cells in the meibomian gland, and was preferentially located adjacent the vacuolated cytoplasm. In culture, the meibocytes began to store lipid droplets in the cytoplasm as they became confluent, and the immunoreactivity for ADRP was found at the margins of the oil droplets. Our results suggest that ADRP can serve as a new marker for the identification of differentiated meibocytes containing lipid droplets.     

Reduction of hepatosteatosis and lipid levels by an adipose differentiation-related protein antisense oligonucleotide

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive triglyceride accumulation in hepatocytes. Expression of the lipid droplet protein adipose differentiation-related protein (ADRP) is increased in NAFLD, but whether this is causally linked to hepatic lipid metabolism is unclear. We postulated that a reduction in ADRP would ameliorate hepatic steatosis and improve insulin action. METHODS: Leptin deficient Lep(ob/ob) and diet-induced obese (DIO) mice were treated with antisense oligonucleotide (ASO) against ADRP, and effects on hepatic and serum lipids and glucose homeostasis were examined. RESULTS: ADRP ASO specifically decreased ADRP mRNA and protein levels in the livers of Lep(ob/ob) and DIO mice, without altering the levels of other lipid droplet proteins, that is, S3-12 and TIP47. ADRP ASO suppressed expression of lipogenic genes, reduced liver triglyceride content without affecting cholesterol, attenuated triglyceride secretion, and decreased serum triglyceride and alanine aminotransaminase levels. The reduction in hepatic steatosis by ADRP ASO was associated with improvement in glucose homeostasis in both Lep(ob/ob) and DIO mice. CONCLUSIONS: This study demonstrates a crucial role for the lipid droplet protein ADRP in regulation of lipid metabolism. Reduction in hepatic ADRP level using an antisense oligonucleotide reverses hepatic steatosis, hypertriglyceridemia, and insulin resistance in obese mice, suggesting that ADRP may be targeted for the treatment of NAFLD and associated lipid and glucose abnormalities.