Differentiation Of Pig Preadipocytes Research Articles

MiR-10a-5p Regulates the Proliferation and Differentiation of Porcine Preadipocytes Targeting the KLF11 Gene.

In the swine industry, meat quality, color, and texture are influenced by the excessive differentiation of fat cells. miRNAs have emerged as integral regulators of adipose development. This study delves into the influence of miR-10a-5b on the proliferation and differentiation of pig preadipocytes. Our findings reveal that miR-10a-5b is prevalent across various tissues. It hinders preadipocyte proliferation, amplifies the expression of adipogenic genes, promotes lipid accumulation, and, as a result, advances preadipocyte differentiation. We predict that KLF11 is the target gene of miRNA. A dual-fluorescence reporter assay was conducted to validate the binding sites of miR-10a-5b on the 3'UTR of the KLF11 mRNA. Results showed that miR-10a-5b targeted KLF11 3'UTR and reduced the fluorescence activity of the dual-fluorescent reporter vector. Our research also indicates that miR-10a-5b targets and downregulates the expression of both mRNA and the protein levels of KLF11. During the differentiation of the preadipocytes, KLF11 inhibited adipose differentiation and was able to suppress the promotion of adipose differentiation by miR-10a-5b. This underscores miR-10a-5b's potential as a significant regulator of preadipocyte behavior by modulating KLF11 expression, offering insights into the role of functional miRNAs in fat deposition.

The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

콜라비가 돼지 지방전구세포와3T3-L1cell의 증식과 분화에 미치는 영향

본 연구는 콜라비가 돼지 지방전구세포와 <TEX>$_3T_3-L_1$</TEX> 세포의 증식과 분화에 미치는 영향을 구명하기 위해 수행하였다. 돼지 지방전구세포는 신생자돈의 등지방에서 분리했다. 세포를 접종한 1일 후에 세척했고(day 0), 세포증식에 미치는 영향을 구명하기 위해서 2일 동안(day 0~day 2) 25 ng/ml과 100 ng/ml의 콜라비 알코올 추출물(과피와 과육)를 처리했다. 세포분화를 구명하기 위해서는 DMEM/F- 12 배지에 6일 동안(day 0~day 6) 배양하고 배양초기 2일 동안(day 0~day 2) 콜라비를 처리하고 day 6에 세포 분화를 측정했다. 콜라비 과피 25 ng/ml와 100 ng/ml은 돼지 지방전구세포의 증식을 각각 4.59%, 17.7% 억제했고, 콜라비 과육은 각각 11.4%, 19.2% 억제했다. 반면 돼지 지방전구세포의 분화는 억제하지 않았다. 콜라비가 <TEX>$_3T_3-L_1$</TEX> cell의 증식과 분화에 미치는 작용을 구명하기 위해, 돼지 지방전구세포처럼, 세포 배양초기 2일간 콜라비를 처리했는데 콜라비 과피와 과육 둘 다 세포의 증식과 분화에 영향을 미치지 않았다. 본 연구의 결과를 요약하면, 콜라비는 돼지 지방전구세포의 증식을 억제했으나 분화는 억제하지 않았고, 한편 <TEX>$_3T_3-L_1$</TEX> cell의 증식과 분화 모두 영향을 미치지 않았다. The current study was carried out to determine the effects of Kohlrabi (Brassica oleracea var. gongylodes) on proliferation and differentiation of pig preadipocytes and <TEX>$_3T_3-L_1$</TEX> cells. Pig preadipocytes were isolated from the backfat of the new-born pigs. Twenty-four hours after seeding, the cells were washed with DMEM/F-12 (designated day 0). To measure the cell proliferation, the cells were treated with 25 ng/ml and 100 ng/ml ethanol extracts of Kohlrabi (peel and flesh) for two days (day 0 ~ 2). To measure differentiation, the cells were treated with Kohlrabi for two days (day 0 ~ 2) and cell differentiation was measured on day 6. Twenty-five ng/ml and 100 ng/ml of Kohlrabi peel decreased proliferation of pig preadipocytes by 4.59% and 17.7%, respectively, compared with the control and Kohlrabi flesh by 11.4% and 19.2%, respectively. However, Kohlrabi did not inhibit cell differentiation. To measure the effects of Kohlrabi on proliferation and differentiation of <TEX>$_3T_3-L_1$</TEX> cells, the cells were treated with Kohlrabi for two days in culture, like pig preadipocytes. Kohlrabi (both peel and flesh) did not show any effects on cell proliferation and differentiation. In summary, the results of the current study showed that Kohlrabi decreased proliferation of pig preadipocytes, but no inhibitory effects on differentiation of the cells. Kohlrabi had no effects on proliferation and differentiation of <TEX>$_3T_3-L_1$</TEX> cells.

한국 약용식물 추출물이 In vitro 돼지 지방조직의 지방합성과 지방전구세포의 분화에 영향을 미친다

In vivo 실험에 의해서 지방축적을 억제할 수 있는 물질을 찾아내는 것은 시간과 비용이 많이 든다. 본 연구는 돼지 지방조직을 이용한 in vitro system을 이용해서 한국의 약용식물 중에서 지방축적 억제 작용을 가지는 것을 조사하고자 수행하였다. 총 183종의 약용식물을 이용하여 이들이 돼지 지방조직의 지방합성과 지방전구세포의 분화에 미치는 작용을 조사하였다. 에탄올 추출물은 72종, 물 추출물은 111종, 이 중에서 65종은 물과 에탄올 모두 이용해서 추출하였다. 돼지 지방조직의 지방합성은 13종류의 약용식물이 영향을 미쳤는데, 그 중 11종은 대조구에 비하여 지방합성을 40% 이상 억제하였고, 4종은 지방합성을 70% 이상 억제하였다. 가장 강력한 지방합성 억제작용을 나타낸 것은 붓꽃과와 고삼(에탄올 추출) 그리고 좁쌀풀(물과 에탄올 추출)이었다. 그러나 소목과 황백은 지방합성을 촉진하였다. 돼지 지방전구세포의 분화에 미치는 결과는 총 28종의 약용식물이 돼지 지방전구세포의 분화에 영향을 미쳤는데, 이 중 16종의 약용식물은 증가시켰고, 12종은 억제시켰다. 목단피와 강활(에탄올 추출) 그리고 당귀, 목향 및 신이(물 추출)는 지방세포의 분화를 두 배 정도 증가시켰다. 10종의 약용식물 즉 감초, 형개 및 구월나무(에탄올 추출) 그리고 비름과, 천문동, 백출, 유자나무, 향부자, 구엽초 및 목단피(물 추출)은 지방세포의 분화를 35% 이상 억제시켰다. 구월나무(에탄올 추출)만이 지방합성뿐만 아니라 세포분화도 억제하였다. 본 연구 결과는 약용식물로부터 가축과 사람의 지방축적 억제효능 후보물질을 발굴하기 위한 기초자료로 활용할 수 있을 것으로 여겨진다. Identification of natural compounds that can prevent the development of obesity in vivo is time consuming and expensive. We have used in vitro systems derived from pig adipose tissue to screen simple aqueous or ethanolic extracts of Korean medicinal herbs (KMH) for their anti-adipogenic potential. A total of 183 extracts were tested for their actions in lipogenesis of pig adipose tissue and differentiation of pig preadipocytes. Ethanol extracts were prepared from 72 and aqueous extracts were prepared from 111 medicinal herbs. Both an ethanolic and an aqueous extract were prepared from 65 of these. Thirteen extracts substantially altered rates of lipogenesis in vitro. The effects of KMH on lipogenesis of pig adipose tissue are as follows. Elevens reduced lipogenesis to rates that were more than 40% lower than control and four of these reduced rates of lipogenesis by more than 70%. The most potent anti-lipogenic extracts were those obtained in ethanol from Iridaceae and from Sophora flavescens AIT as well as both the aqueous and ethanolic extracts from Lysimachia vulgaris L. Two extracts, those prepared in water from Caesalpiniae lignum and from Phellodendri cortex, were found to promote rates of lipogenesis in vitro. The effects of KMH on differentiation of pig preadoipocytes are as follows. Twentyeight extracts altered the rates of differentiation of cultured porcine preadipocytes. Sixteen increased and twelve reduced the rates of differentiation of preadipocytes. Extracts prepared in ethanol from Moutan radicis cortex and from Ostericum koreanum and those prepared in water from Angelicae gigantis radix, from Inula henenium L and from Magnolia flos doubled the rate of differentiation of cultured porcine preadipocytes. Ten extracts reduced the in vitro rate of differentiation of porcine preadipocytes by more than 35%. These were the ethanolic extracts from Glycyrrizae radix, Nepetae spica and from Polygala myrtifolia and the aqueous extracts from Amaranthaceae, Asparagus cochinchinesis, Atractylodis rhizoma alba, Citrus junos TANAKA, Cyperus rotundus, Epimedium grandiflorum and from Moutan radicis cortex. Only the ethanolic extract from Polygala myrtifolia was able to both reduce lipogenesis in adipose tissue slices and retard differentiation of cultured preadipocytes. The results of our study will provide meaningful information to identify medicinal herbs which would reduce fat deposition in livestocks and humans.

Retinoic Acid가 돼지 지방세포의 분화에 미치는 영향

Retinoic Acid가 돼지 지방세포의 분화에 미치는 영향

Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes

Sirt1, a NAD(+)-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARgamma2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.

Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids1

Conjugated linoleic acids are a group of geometric and positional isomers of linoleic acid that decrease body fat in growing animals by a poorly understood mechanism. The objective of this study was to investigate the isomer-specific effect of CLA on the proliferation and differentiation of pig preadipocytes in primary culture. The effect of CLA on preadipocyte proliferation was determined using cleavage of the tetrazolium salt, WST-1, as a marker for proliferation. Preadipocyte number was decreased in a dose-dependent fashion by trans-12,cis-10 CLA (P < 0.05). No other fatty acid affected preadipocyte number. Differentiation was monitored on d 10 after induction morphologically, enzymatically, and by measuring the mRNA abundance of key adipogenic transcription factors. Both a crude CLA preparation containing a mixture of CLA isomers (CLA-mix) and the pure trans-10,cis-12 CLA isomer inhibited glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent fashion, with trans-10,cis-12 CLA being more potent (P < 0.01) than the CLA-mix. Cis-9,trans-11 CLA failed to decrease GPDH activity; however, increasing concentrations of cis-9,trans-11 CLA tended to blunt the inhibitory effect of trans-10,cis-12 CLA on GPDH activity (P < 0.09), suggesting that cis-9,trans-11 CLA may antagonize the action of trans-10,cis-12 CLA in porcine adipocytes. Finally, the isomer-specific effect of CLA on adipogenic transcription factor gene expression was investigated. Trans-10,cis-12 CLA decreased expression of peroxisome proliferator-activated receptor gamma (PPAR gamma; P < 0.01) and sterol regulatory element-binding protein-1c (SREBP-1c; P < 0.05) mRNA, while failing to alter the expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) mRNA. Interestingly, both the CLA-mix and the trans-10,cis-12 CLA isomer increased the mRNA abundance of chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF; P < 0.002). No other fatty acid affected COUP-TF mRNA levels. Collectively these data support the concept that CLA decreases fat accretion in pigs, in part by inhibiting preadipocyte proliferation and differentiation, with trans-10,cis-12 CLA being an active isomer eliciting these effects. Furthermore, trans-10,cis-12 CLA inhibits porcine preadipocyte differentiation by a mechanism that involves the down-regulation of PPARgamma and SREBP-1c mRNA. This mechanism is independent of changes in C/EBPalpha mRNA abundance and may involve COUP-TF.