Differential effects of bariatric surgery and caloric restriction on hepatic one-carbon and fatty acid metabolism

Summary: Weight loss interventions, including dietary changes, pharmacotherapy, or bariatric surgery, prevent many of the adverse consequences of obesity, and may also confer intervention-specific benefits beyond those seen with decreased weight alone. We compared the molecular effects of different interventions on liver metabolism to understand the mechanisms underlying these benefits. Male rats on a high-fat, high-sucrose diet underwent sleeve gastrectomy (SG) or intermittent fasting with caloric restriction (IF-CR), achieving equivalent weight loss. The interventions were compared to ad-libitum (AL)-fed controls. Analysis of liver and blood metabolome and transcriptome revealed distinct and sometimes contrasting metabolic effects between the two interventions. SG primarily influenced one-carbon metabolic pathways, whereas IF-CR increased de novo lipogenesis and glycogen storage. These findings suggest that the unique metabolic pathways affected by SG and IF-CR contribute to their distinct clinical benefits, with bariatric surgery potentially influencing long-lasting changes through its effect on one-carbon metabolism

The expression of the beta cell-derived autoimmune ligand for the killer receptor nkp46 is attenuated in type 2 diabetes.

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D

Recovery of NKp46 ligand after reconstitution of leptin in ob/ob mice.

<p>Mice (ob/ob) were injected I.V with an adeno-vector encoding for GFP (Adeno GFP, A-C) or with an adeno-vector encoding for murine leptin (B-C). (B-C) Immunohistochemical (B) and Immunofluorescence (C) NKp46-Ig staining of pancreatic tissue derived from 8–12 weeks male ob/ob mice treated for 14 days with adeno-vector encoding for leptin (B, right and C lower) or with adeno-vector encoding for GFP (B, left and C upper). (C) Left images represent staining with NKp46-Ig (NKp46 ligand, green), middle images represent staining with anti-insulin antibody (Insulin, red) and the right images represent merge signal (Merge, yellow). (A and C) Magnificationx400, scale bar-50 µm, (B) Magnificationx200, scale bar-25 µm. Representative of two independent experiments, 3–4 mice in each group.</p

Insulinoma staining.

<p>(A-B) Immunofluorescence images of INS-1E (A) and MIN6 (B) beta cell lines. Left images represent staining with anti-insulin antibody (Insulin, red), middle images represent staining with the NKp46-Ig (NKp46 ligand, green) and the right images represent the merge signal (Merge, yellow). Arrows indicate areas positive for NKp46-Ig staining and negative for insulin. Representative of three independent staining is shown. Magnificationx1800, scale bar-10 µm.</p

Expression of insulin and NKp46 ligand in T2D models.

<p>(A) Immunohistochemical staining of pancreatic tissues derived from 8–10 weeks old control C57BL/6 mice (top), 8–10 weeks old leptin deficient (OB/OB) mice (middle) and 5–6 weeks old leptin receptor deficient (DB/DB) mice (bottom). Staining was performed with NKp46-Ig. Magnificationx400. Representative of three independent staining is shown. Tissues were obtained from 3–4 mice in each group. (B) Immunofluorescence staining of pancreatic tissues derived from 8–10 weeks old control C57BL/6 mice (top), 8–10 weeks old OB/OB mice (middle) and 5–6 weeks old DB/DB mice (Bottom). Left images represent staining with NKp46-Ig (NKp46 ligand, green), middle images represent staining with anti-insulin (Insulin, red) and the right images represent the merge signal (Merge, yellow). Magnificationx400. Scale bars-50 µm. Representative of three independent staining is shown, 3–4 mice in each group.</p

NKp46 does not bind to insulin.

<p>(A) Plates were coated with the indicated Ig-fusion proteins (NKp46-Ig and NKp30-Ig, X axis) and then incubated either with biotinylated antibodies (α-NKp46, and α-CD3) or with biotinylated insulin (Ins). ELISA detection was performed using Streptavidin-HRP. (B) FACS analysis for the expression of NKp46 on parental BW cells (top) and on NKp46-CD3-zeta (bottom) transfected BW cells. (C) NKp46-CD3-zeta transfected BW cells were incubated with insulin, glucagon or BSA, in the presence (+721.221Un) or in the absence of 721.221 cells. In addition, 721.221 cells infected with PR8 influenza (+721.221In) were incubated with NKp46-CD3-zeta transfected BW cells. IL-2 was detected in the supernatants using standard ELISA.</p

Electron microscopy images of NKp46 ligand and of insulin.

<p>INS-1E cells were co-stained with NKp46-Ig (upper, large black dots, 15 nm) and KIR2DS4-Ig (lower) together with anti-insulin (small black dots, 6 nm).</p

Stimulation of insulin secretion increases the expression of the NKp46 ligand.

<p>(A-B) INS-1E cells were incubated for different periods of time (indicated in the Figure) in Krebs-Ringer bicarbonate HEPES–BSA buffer containing 3.3 mM and 16.7 mM glucose. Cells were harvested at each time point and NKp46 ligand expression was analyzed by cytospin (A) and by flow cytometry (B). Shown in (A) is the intensity (colors intensity is indicated in the right of the Figure) of the expression of NKp46 ligand in INS-1E cells using the NKp46-Ig. MagnificationX400, scale-50 µm. (B) Flow cytometry staining for the surface expression of the NKp46 ligand in INS-1E cells following stimulation at 16.7 mM glucose (green histograms-unstimulated cells, red-stimulated). The MFI values are indicated in the corresponding histograms. (C) INS-1E cells shown in A were incubated at 16.7 mM glucose for different periods of time and secreted insulin was analyzed by an ELISA assay. Results are expressed as microgram/liter (mic/l). Error bars (SD) are derived from triplicates. Data from one of three independent assays is shown.*p = 0.0125 (0/10min), **p = 0.014 (10/30min). (D) ELISA assays for the detection of the NKp46 ligand in the supernatants of the INS-1E cells shown in (A) before and during glucose stimulation. Data from one of two independent assays is shown.</p