Beneficial effects of Brazilian propolis on type 2 diabetes in ob/ob mice: Possible involvement of immune cells in mesenteric adipose tissue

Abstract

The anti-diabetic effects of Brazilian propolis were examined using ob/ob mice. Although repeated injection of an ethanol extract of Brazilian propolis (100 mg/kg, ip, twice a week for 12 weeks) did not affect body weight gain and food intake of ob/ob mice, blood glucose and plasma cholesterol levels were significantly attenuated. Moreover, the propolis extract partially restored glucose tolerance and insulin resistance, indicating anti-diabetic properties of the extract. The propolis-treated mice exhibited lower weight gain in mesenteric adipose tissue, while weight gains in inguinal and epididymal adipose tissues were not modulated. Flow cytometric and microscopic analyses suggested that the extract promoted accumulation of eosinophils into mesenteric and epididymal adipose tissues. Alternatively, the ratio of M1-like macrophages to M2-like macrophages in mesenteric adipose tissue was reduced by the propolis injection, coincident with the decrement of the number of interleukin-12A(+) cells. Levels of M1 macrophage markers, such as Itgax and Il12b transcripts, were decreased in the vascular stromal fraction of mesenteric adipose tissue, whereas those of pan-macrophage markers Emr1 and Cd68 were not influenced. Microarray and subsequent gene ontology term analyses suggested that propolis attenuated immune activation in mesenteric adipose tissues. Taken together, this indicates that Brazilian propolis improves diabetes in ob/ob mice, presumably through modification of immune cells in mesenteric adipose tissues.

Keywords: adipose tissue macrophage; chronic inflammation; eosinophils; metaflammation; ob/ob mouse; propolis; type 2 diabetes.

Figure 1. Effects of intraperitoneal injection of Brazilian propolis on metabolic states of ob/ob mice. (A) Effects of a Brazilian propolis ethanol extract on body weight (left) and food intake (right) in ob/ob mice. Brazilian propolis ethanol extract (100 mg/kg, ip, twice a week) or vehicle was injected into ob/ob mice for 12 weeks. Values are the mean ± SD of 9 mice. The P values calculated by two-way repeated measures ANOVA are also shown. (B) Casual plasma glucose and lipid levels. Following 12 weeks of propolis injection, plasma was collected and subjected to analyses. Values are the mean ± SD of 9 mice. *P < 0.05 vs vehicle treated mice. (C) Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Following 12–13 weeks of propolis injection, mice were subjected to OGTT and ITT. Values are the mean ± SD of 6 (OGTT) and 11 (ITT) mice. The P values calculated by two-way repeated-measures ANOVA are also shown. (D) Wet tissue weight of white adipose tissues (WAT). Following 12 weeks of propolis injection, inguinal, epididymal, and mesenteric adipose tissues were collected. Values are the mean ± SD of 9–10 mice. *P < 0.05 vs the vehicle-treated mice.

Figure 2. Effects of intraperitoneal injection of a Brazilian propolis ethanol extract on the proportion of eosinophils and macrophages in epididymal adipose tissue of ob/ob mice. A Brazilian propolis ethanol extract (100 mg/kg, ip, twice a week) or vehicle was injected into ob/ob mice for 12 weeks. The vascular stromal fraction (VSF) of epididymal adipose tissue was used for FACS analysis. (A) Representative images of FACS analysis of eosinophils and macrophages. After removal of dead cells, eosinophils and macrophages were defined by the staining pattern of Siglec-F and CD11b. (B) The proportion of eosinophils and macrophages in living cells in the VSF of epididymal adipose tissue. Values are the mean ± SD of 7 mice. *P < 0.05 vs the vehicle-treated mice.

Figure 3. Effects of intraperitoneal injection of a Brazilian propolis ethanol extract on the proportion of eosinophils, macrophages, and total lymphocytes in the mesenteric adipose tissue of ob/ob mice. A Brazilian propolis ethanol extract (100 mg/kg, ip, twice a week) or vehicle was injected into ob/ob mice for 12 weeks. (A) Representative images of FACS analysis of eosinophils and macrophages in the VSF of mesenteric adipose tissue. After removal of dead cells, eosinophils and macrophages were defined by the staining pattern of Siglec-F and CD11b. (B) The proportion of immune cells in living cells in the VSF of mesenteric adipose tissue. Total lymphocytes were defined by forward scatter (FSC) and side scatter (SSC). Values are the mean ± SD of 7 mice. *P < 0.05 vs the vehicle-treated mice. (C) Hematoxylin and eosin staining of mesenteric adipose tissue. Representative images of three experimental series are shown. An enlarged view of the propolis-treated sample is also shown. An arrowhead indicates an eosinophil. Bars represent 20 μm.

Figure 4. Effects of a Brazilian propolis ethanol extract on subpopulations of macrophages and T lymphocytes. A Brazilian propolis ethanol extract (100 mg/kg, ip, twice a week) or vehicle were injected into ob/ob mice for 12 weeks. (A) Effects of propolis on the proportion of macrophage subpopulations. (Left) Representative FACS images of CD11c⁺ M1-like macrophages and CD206⁺ M2-like macrophages in mesenteric adipose tissue. The percentage proportions of each macrophage subpopulation in F4/80⁺ cells are also shown. (Right) The ratio of M1-like macrophages to M2-like macrophages. Values are the mean ± SD of 7 mice. *P < 0.05 vs the vehicle-treated mice. (B) Imunohistochemical detection of M1-like and M2-like macrophages in mesenteric adipose tissue. M1-like and M2-like macrophages were detected using antibodies for IL-12A and Mgl1/2, respectively. Arrowheads indicate immunosignals. Scale bars represent 100 μm. (C) Effects of proportion of T-lymphocyte subpopulations. (Left) Representative FACS images of CD4⁺ CD8⁻ helper T lymphocytes and CD4⁻ CD8⁺ cytotoxic T lymphocytes in mesenteric adipose tissue. The percentage proportions of each T-lymphocyte populations are also shown. (Right) The ratio of cytotoxic T lymphocytes to helper T lymphocytes (8T/4T ratio). Values represent the mean ± SD of 7 mice. WAT, white adipose tissue.

Figure 5. Effects of a Brazilian propolis ethanol extract on the transcriptome of the VSF of mesenteric adipose tissue. A Brazilian propolis ethanol extract (100 mg/kg, ip, twice a week) or vehicle were injected into ob/ob mice for 12 weeks. Triplicate total RNA samples were prepared from the VSF of mesenteric adipose tissue, and subjected to a microarray analysis. (A) Heatmap representation of expression of the propolis-affected genes. Numbers of increased and decreased genes were 519 and 205 genes, which corresponded to 730 and 277 probes, respectively. Clustering trees are also depicted. (B) Gene Ontology (GO) term analysis of the propolis-attenuated genes using the GOrilla program. We selected Biological Process ontology terms for this analysis. Terms and the P value are shown. Terms associated with immune responses are noted in blue. (C) A scatter plot representation of GO analysis data by the REViGO program. The scatter plot shows a two dimensional cluster representative of the Biological Process ontology terms’ semantic similarities. The color of the circles represents the P value; size indicates the frequency of the GO term in the Gene Ontology Annotation data set. (D) An annotation cluster of the propolis-attenuated genes. Functional clustering of genes and annotation terms were performed by the DAVID program. A cluster showing high enrichment score includes immune activation genes. Green areas indicate the term–gene pairs.

Figure 6. qRT-PCR analysis of macrophage molecules using the mesenteric adipose tissues of the propolis-treated ob/ob mice. A Brazilian propolis ethanol extract (100 mg/kg, ip, twice a week) or vehicle was injected into ob/ob mice for 12 weeks. Total RNA isolated from the VSF of mesenteric adipose tissue was subjected to qRT-PCR analysis. Expression of (A) Pan, M1, and M2 marker genes, and (B) genes participating in pathogenesis of atherosclerosis were measured. Values are normalized to Hprt1 mRNA levels and represented as the mean ± SD of 4–5 mice. Log10-transformed values were subjected to the Student t test. *P < 0.05 vs vehicle treated mice. All primers used are listed in Table S1.