Location, location, location: Beneficial effects of autologous fat transplantation

Abstract

Visceral adiposity is a risk factor for cardiovascular disorders, type 2 diabetes mellitus (T2D) and associated metabolic diseases. Sub-cutaneous fat is believed to be intrinsically different from visceral fat. To understand molecular mechanisms involved in metabolic advantages of fat transplantation, we studied a rat model of diet-induced adiposity. Adipokine genes (Adiponectin, Leptin, Resistin and Visfatin) were expressed at 10,000 to a million-fold lower in visceral fat depot as compared to peripheral (thigh/chest) fat depots. Interestingly, autologous transplantation of visceral fat to subcutaneous sites resulted in increased gene transcript abundance in the grafts by 3 weeks post-transplantation, indicating the impact of local (residence) factors influencing epigenetic memory. We show here that active transcriptional state of adipokine genes is linked with glucose mediated recruitment of enzymes that regulate histone methylation. Adipose depots have "residence memory" and autologous transplantation of visceral fat to sub-cutaneous sites offers metabolic advantage.

Figure 1. Adipokine gene expression in fat depots.

(A) Adipose tissue from different sites was snap frozen and collected for gene expression analysis using TaqMan-based quantitative real-time PCR. (B) Expression of Adipoq (Adiponectin), Lep (Leptin), Retn (Resistin) and Nampt (Visfatin) were significantly lower in visceral adipose tissue (red circles) as compared to adipose tissue obtained from other (peripheral) sites / depots. Gene expression for each animal is shown in the scatter plot (N = 9).

Figure 2. Adipokine gene expression in Visceral fat depot matches expression levels in resident (thigh) depot following autologous transplantation.

Following 3 weeks after transplantation of visceral adipose tissue (V) to subcutaneous (thigh) region (T) the abundance of adipokine gene transcripts in the transplanted (VT) tissue was estimated by TaqMan-based quantitative PCR. We observe that gene transcript abundance in the grafts (VT) was significantly higher than that observed on the day of transplantation (Visceral;V), and not different from the abundance of gene transcript detected in the resident thigh fat.

Figure 3. Adipokine gene promoters in visceral fat acquire active marks (H3K4 methylation) following translocation of visceral fat to peripheral (thigh/chest) sites.

Chromatin immunoprecipitation (ChIP) followed by quantitative real-time PCR was carried out using promoter-specific probe-primers for each adipokine promoter region. Data are presented relative to methylation at promoter region of visceral fat (V) isolated at time of transplantation. We assessed H3K4 (A) and H3K9 (B) dimethylation as active and inactive marks of gene expression respectively. Following fat translocation to thigh (T) or chest (C) region, a significant increase in H3K4 methylation was observed for almost all the adipokine gene promoter regions.

Figure 4. Changes in adipokine promoter methylation involve recruitment of LSD1 and KMT1a.

LSD1 and KMT1a recruitment at promoter regions is suggestive of gene repression (see text). Recruitment of LSD1 and KMT1a at adipokine gene promoter regions was assessed by carrying out chromatin immunoprecipitation for the 2 enzymes. Either LSD1 and/or KMT1a abundance at adipokine promoters was seen to decrease significantly following transplantation of visceral fat (V) to Thigh (VT) or chest (VC) region.

Figure 5. Autologous transplantation of fat offers metabolic advantage.

To confirm any metabolic advantages offered as a result of autologous fat transplantation procedure, we measured circulating concentrations of (A) adiponectin, (B) leptin and (C) endotoxin in serum of rats before (day 0) or after (day 21) surgery. Hyperinsulinemic euglycemic clamps were carried out (D,E) to confirm that translocation of visceral fat to sub-cutaneous sites offered improved insulin sensitivity in these rats. P<0.001, N = 5.

Figure 6. Glucose alone can induce increase in adipokine gene expression.

Blood glucose measurements were simultaneously carried out at 30 minutes after oral glucose was administered (A). We observe that local concentration of glucose in portal circulation are higher before first pass through liver, N = 6♂,7♀(B). Rats generally eat throughout the day/night and visceral adipose tissue is therefore exposed to high concentrations of glucose as compared to peripheral adipose tissue. To test the role of glucose, we harvested visceral fat from adult rats and exposed it to low glucose conditions for 72 hours (C). Following exposure to low glucose conditions, adiponectin, leptin, resistin and visfatin mRNA increased by at least 200-fold as compared to the abundance of these transcripts in the harvested (day 0) visceral tissue (N = 4 rats).

Figure 7. Analysis of glucose-regulated genes.

Taqman low density array (TLDA) analysis of gene expression in visceral (V) and transplanted fat (VT) in comparison to thigh fat (T) was carried out for several genes that are known to be involved in the metabolic syndrome. Two distinct groups of genes were observed; (A) glucose-responsive genes (N = 9; 14 genes): These were influenced as a result of transplantation to peripheral site, while other (B) glucose non-responsive or site independent genes (N = 9, 18 genes) did not show significant change in gene expression. (Adipor1: Adiponectin receptor 1, Adipor2: Adiponectin receptor 2, Hand1: Heart and neural crest derivatives expressed 1, Npy: neuropeptide Y, Ahcy: adenosylhomocysteinase, Dhfr: Dihydrofolate reductase, Sgpl1: sphingosine phosphate lyase 1, Irs1: Insulin receptor substrate 1, Faah: Fatty acid amide hydrolase, Cubn: Cubilin, Crp: C-reactive protein, Lpl: Lipoprotein lipase, Lrp2: Low density lipoprotein receptor-related protein 2, Prkaa2: protein kinase, AMP-activated, alpha 2 catalytic subunit, Mal2a: mal, T-cell differentiation protein 2, Mtr: 5-methyltetrahydrofolate-homocysteine methyltransferase, Nfkb: Nuclear factor kappa-B, Calca: Calcitonin related polypeptide alfa, Hmbs: hydroxymethylbilane synthase, Fto: Fat mass and obesity associated, Pparg: peroxisome proliferator-activated receptor gamma, Srebf1: sterol regulatory element binding transcription factor 1, Agrp: Agouti related protein homolog, Irs2: Insulin receptor substrate 2, Tcn2: transcobalamin 2, Rela: v-rel reticuloendotheliosis viral oncogene homolog A, FoxP1: Forkhead box P1, Prkag2: protein kinase, AMP-activated, gamma 2 non-catalytic subunit, Cnr1: cannabinoid receptor 1 (brain), Fabp4: fatty acid binding protein 4, adipocyte, Atp2a1: Ca++ transporting, cardiac muscle, fast twitch 1, Bmpr2: Bone morphogenetic protein receptor 2)