Role of IGFBP-3 in the Regulation of β-Cell Mass during Obesity: Adipose Tissue/ β-Cell Cross Talk

In obesity an increase in β-cell mass occurs to cope with the rise in insulin demand. This β -cell plasticity is essential to avoid the onset of hyperglycemia, although the molecular mechanisms that regulate this process remain unclear. This study analyzed the role of adipose tissue in the control of β -cell replication. Using a diet-induced model of obesity, we obtained conditioned media from three different white adipose tissue depots. Only in the adipose tissue depot surrounding the pancreas did the diet induce changes that led to an increase in INS1E cells and the islet replication rate. To identify the factors responsible for this proliferative effect, adipose tissue gene expression analysis was conducted by microarrays and quantitative RT-PCR. Of all the differentially expressed proteins, only the secreted ones were studied. IGF binding protein 3 (Igfbp3) was identified as the candidate for this effect. Furthermore, in the conditioned media, although the blockage of IGFBP3 led to an increase in the proliferation rate, the blockage of IGF-I receptor decreased it. Taken together, these data show that obesity induces specific changes in the expression profile of the adipose tissue depot surrounding the pancreas, leading to a decrease in IGFBP3 secretion. This decrease acts in a paracrine manner, stimulating the β -cell proliferation rate, probably through an IGF-I-dependent mechanism. This cross talk between the visceral-pancreatic adipose tissue and β -cells is a novel mechanism that participates in the control of β -cell plasticity. (Endocrinology 153: 177–187, 2012) Adult male Wistar rats (Charles River Laboratories, Wilmington, MA), 7 wk old (weighing 225–250 g), were caged individually in a 12-h light, 12-h dark cycle in a temperature- and humidity-controlled environment. Animals were divided into two dietary sets for 30 days. One group was fed with standard chow diet (supplying 8% of calories as fat; type AO4 from Panlab, Barcelona, Spain). The second group was fed with a cafeteria diet (66% of calories as fat), as previously described (Endocrinology 146:4362–4369, 2005). Adipose tissue from the mesenteric surrounding the pancreas (pMES), was excised, weighed, cut, and rapidly frozen in liquid nitrogen for RNA isolation. Ten micrograms of total RNA from pMES adipose tissue were converted into cRNA, biotinylated, fragmented, and hybridized to GeneChip Rat Genome 230 2.0 (Affymetrix, Santa Clara, CA). Five microarrays were hybridized, three with independent samples coming from rats fed with standard chow (lean group) and two with independent samples coming from rats fed with the cafeteria diet (obese group).

在肥胖状态下，机体为应对胰岛素需求的升高，会出现β细胞质量（β-cell mass）的增加。这种β细胞可塑性对于避免高血糖（hyperglycemia）的发生至关重要，尽管调控这一过程的分子机制目前仍不明确。本研究分析了脂肪组织在调控β细胞复制中的作用。我们利用饮食诱导的肥胖模型，从三种不同的白色脂肪组织位点中获取了条件培养基（conditioned media）。仅在胰腺周围的脂肪组织位点中，高脂饮食诱导的变化可使INS1E细胞数量及胰岛增殖率（islet replication rate）升高。为鉴定介导这种增殖效应的因子，我们通过微阵列（microarrays）与实时定量逆转录聚合酶链式反应（quantitative RT-PCR）开展了脂肪组织基因表达分析。在所有差异表达的蛋白中，仅对分泌型蛋白进行了研究。胰岛素样生长因子结合蛋白3（IGF binding protein 3, IGFBP3）被鉴定为该效应的候选因子。此外，在条件培养基中，阻断IGFBP3会使增殖率升高，而阻断胰岛素样生长因子I受体（IGF-I receptor）则会降低增殖率。综合来看，这些数据表明，肥胖会诱导胰腺周围脂肪组织位点的表达谱发生特异性改变，进而降低IGFBP3的分泌。这种分泌减少以旁分泌（paracrine）方式发挥作用，通过依赖于胰岛素样生长因子I（IGF-I）的机制刺激β细胞增殖。这种内脏胰腺旁脂肪组织与β细胞之间的交叉对话是调控β细胞可塑性的全新机制。（《内分泌学（Endocrinology）》153卷：177–187，2012年）

成年雄性Wistar大鼠（购自查尔斯河实验室（Charles River Laboratories，美国特拉华州威尔明顿）），7周龄，体重225–250g，于12小时光照/12小时黑暗循环、温度与湿度可控的环境中单笼饲养。将动物随机分为两组，进行为期30天的膳食干预：一组饲喂标准啮齿动物饲料（脂肪供能占比8%，型号AO4，购自西班牙巴塞罗那Panlab公司）；另一组饲喂自助式高脂饲料（脂肪供能占比66%），实验方案参照此前发表的研究（《内分泌学（Endocrinology）》146卷：4362–4369，2005年）。

摘取胰腺周围肠系膜脂肪组织（pMES），称重、切割后迅速置于液氮中冷冻，用于RNA提取。取10μg该脂肪组织的总RNA，反转录为互补RNA（cRNA）并进行生物素标记、片段化，随后与GeneChip大鼠基因组230 2.0芯片（Affymetrix，美国加利福尼亚州圣克拉拉）进行杂交。共完成5次芯片杂交：3次来自饲喂标准饲料的正常体重组（lean组）的独立样本，2次来自饲喂自助式高脂饲料的肥胖组（obese group）的独立样本。