Mice lacking neutral amino acid transporter B⁰AT1 (Slc6a19) have elevated levels of FGF21 and GLP-1 and improved glycaemic control

OBJECTIVE: Type 2 diabetes arises from insulin resistance of peripheral tissues followed by dysfunction of β-cells in the pancreas due to metabolic stress. Both depletion and supplementation of neutral amino acids have been discussed as strategies to improve insulin sensitivity. Here we characterise mice lacking the intestinal and renal neutral amino acid transporter B⁰AT1 (Slc6a19) as a model to study the consequences of selective depletion of neutral amino acids. METHODS: Metabolic tests, analysis of metabolite levels and signalling pathways were used to characterise mice lacking the intestinal and renal neutral amino acid transporter B⁰AT1 (Slc6a19). RESULTS: Reduced uptake of neutral amino acids in the intestine and loss of neutral amino acids in the urine causes an overload of amino acids in the lumen of the intestine and reduced systemic amino acid availability. As a result, higher levels of glucagon-like peptide 1 (GLP-1) are produced by the intestine after a meal, while the liver releases the starvation hormone fibroblast growth factor 21 (FGF21). The combination of these hormones generates a metabolic phenotype that is characterised by efficient removal of glucose, particularly by the heart, reduced adipose tissue mass, browning of subcutaneous white adipose tissue, enhanced production of ketone bodies and reduced hepatic glucose output. CONCLUSIONS: Reduced neutral amino acid availability improves glycaemic control. The epithelial neutral amino acid transporter B⁰AT1 could be a suitable target to treat type 2 diabetes.This work was supported by a sponsored research agreement with Sanofi-Aventis, Germany

Browning of White Adipose Tissue Uncouples Glucose Uptake from Insulin Signaling

<div><p>Presence of thermogenically active adipose tissue in adult humans has been inversely associated with obesity and type 2 diabetes. While it had been shown that insulin is crucial for the development of classical brown fat, its role in development and function of inducible brown-in-white (brite) adipose tissue is less clear. Here we show that insulin deficiency impaired differentiation of brite adipocytes. However, adrenergic stimulation almost fully induced the thermogenic program under these settings. Although brite differentiation of adipocytes as well as browning of white adipose tissue entailed substantially elevated glucose uptake by adipose tissue, the capacity of insulin to stimulate glucose uptake surprisingly was not higher in the brite state. Notably, in line with the insulin-independent stimulation of glucose uptake, our data revealed that brite recruitment results in induction of solute carrier family 2 (GLUT-1) expression in adipocytes and inguinal WAT. These results for the first time demonstrate that insulin signaling is neither essential for brite recruitment, nor is it improved in cells or tissues upon browning.</p></div

Brown adipose tissue harbors a distinct sub-population of regulatory T cells.

Regulatory T (Treg) cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT). Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress

Lack of insulin impairs differentiation but not browning capacity of primary pre-adipocytes.

<p>(<b>A</b>) Heatmap showing differential mRNA expression between confluent primary inguinal white adipose tissue (iWAT) precursor cells differentiated for 24 h with white (EtOH treated) or brite (cPGI₂ treated) differentiation cocktail and between absence or presence of insulin (Ins) in the medium. Higher and lower expression is displayed in red and blue, respectively. (n = 3). (<b>B</b>) mRNA expression of UCP-1 and CIDEA or (<b>C</b>) FABP4 and RETN in primary iWAT precursor cells differentiated into white (EtOH treated) or brite (cPGI₂ treated) adipocytes for 8 days with insulin present in the differentiation medium for the indicated timepoints (n = 3). All values in bar graphs are expressed as means ± SEM, #p<0.05, ##p<0.01, ###p<0.001 white (EtOH treated) vs. brite (cPGI₂ treated) cells, *p<0.05, **p<0.01, ***p<0.001 normal conditions vs. insulin deprived conditions.</p

Brite adipose cells and tissues exhibit elevated glucose uptake independent of insulin stimulation, thereby enhancing glucose clearance from blood.

<p>(<b>A, B</b>) ³H-2-deoxy-D-glucose (3H-2DOG) uptake by primary inguinal white adipose tissue (iWAT) precursor cells (<b>A</b>) absolute or (<b>B</b>) relative to unstimulated basal uptake. Cells were differentiated into white (EtOH treated) or brite (cPGI₂ treated) adipocytes for 8 days and stimulated with different doses of Insulin for 20 minutes. (<b>C, D</b>) Intraperitoneal insulin (Ins) tolerance test (0.5 U/kg body weight insulin) of mice treated with CL316,243 (CL, 1 µg/g/day) or NaCl via s.c. implanted osmotic pumps for 10 days. (<b>C</b>) Absolute blood glucose levels and (<b>D</b>) levels relative to non-insulin-stimulated are shown. (<b>E, F</b>) 3H-2DOG uptake rate into inguinal or abdominal white (iWAT, aWAT) or brown (BAT) adipose tissue and heart of the same mice as in B, C. (<b>E</b>) Absolute uptake and (<b>F</b>) uptake relative to non-insulin-stimulated conditions is shown. Uptake rates were measured 45 minutes after intraperitoneal injection of insulin or vehicle. All values are expressed as means ± SEM, n = 3–6, #p<0.05, ##p<0.01, ###p<0.001 white vs. brite, *p<0.05, **p<0.01, ***p<0.001 no insulin vs. insulin stimulated.</p

Physiological parameters.

<p>(A) Body weight (BW) and (B) adipose tissues weights of T_reg cell-proficient (PBS) and T_reg cell-deficient (DT) mice after cold exposure. BAT, brown adipose tissue; scWAT, subcutaneous white adipose tissue, aWAT, abdominal white adipose tissue. (C) Blood glucose, (D) serum non-estherified fatty acids (NEFA) and (E) serum triglycerides in PBS and DT mice. Values are mean ± SD (n = 9–10); *P<0.05 (Student’s t-test).</p

Principal biological processes affected by T_reg cell depletion in BAT.

<p>Principal biological processes affected by T_reg cell depletion in BAT.</p

Genotypical comparison of T_reg and T_conv cells isolated from brown adipose tissue (BAT) and spleen tissue (SPL) in cold- and warm-conditioned animals generated with an Illumina Mouse Expression Array.

<p>(A) Gene expression profiles comparing T_reg (top) or T_conv (bottom) cell populations between spleen and adipose tissue samples isolated from warm-conditioned animals (left) or between cells isolated from cold vs warm-conditioned animals (right). Numbers indicate genes either up- or downregulated more than 2-fold (cut-off: dotted line), with the number of significantly different (p<0.05) genes shown in brackets with an asterisk. (B) Volcano plot comparing gene expression and significance values between T_reg and T_conv genes isolated from BAT in warm-conditioned animals. Key up- or downregulated genes in T_reg cells are annotated (Foxp3, Il10, Cxcl1/2, Tcf7, Ifng) and serve as quality control to the published consensus T_reg-cell signature. (C) Hierarchical clustering of the top-30 upregulated genes and the top-10 downregulated genes in warm-conditioned brown adipose tissue T_reg cells versus spleen T_reg cells. (D) Comparison of BAT-T_reg-specific genes with visceral adipose tissue (VAT)-specific genes. We first determined 430 genes to upregulated in BAT warm-conditioned T_reg cells, with 222 genes being significantly altered (p<0.05). We then overlaid BAT T_reg-upregulated genes with VAT T_reg tissue specific expression gene data. 181 genes were matched between both microarary chips, with 169 genes also upregulated in VAT, and only 12 genes specific for BAT (left). The corresponding analysis of the 516 genes upregulated in cold BAT T_reg cells versus warm spleen T_reg cells revealed 194 genes to be significantly upregulated. 158 could be matched to VAT T_reg-specific genes, of which 148 were VAT-specific, whereas only 10 were specific for BAT. P-values indicate the significance of overrepresentation of BAT T_reg-specific genes in the VAT T_reg signature. (E) Comparison of VAT-T_reg specific genes on BAT warm (left) or BAT cold (right) gene signatures. Of 1839 genes specifically overexpressed in VAT T_reg cells, 1059 were statistically significantly (p<0.05) upregulated. Of these 1059 genes, 829 were also detectable in the BAT T_reg microarray. When comparing the VAT T_reg signature to warm BAT T_reg cells, 660 genes were overrepresented in VAT, whereas cold BAT T_reg cells show 685 genes to be overrepresented in VAT.</p

Inflammatory status of adipose tissue.

<p>Real-time RT-PCR analysis of (A) brown adipose tissue (BAT) and (B) subcutaneous white adipose tissue of T_reg cell-proficient (PBS) and T_reg cell-deficient (DT) mice after cold exposure. Ucp1, uncoupling protein 1; Cidea,cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; Dio2,deiodinase, iodothyronine, type II; Pparg, peroxisome proliferator-activated receptor gamma; Prdm16, PR domain containing 16; Cd68, Cd68 antigen; Ccl2,chemokine (C-C motif) ligand 2; Tnfa, tumor necrosis factor alpha; Ifng, interferon, gamma; Mrc1, mannose receptor, C type 1; Mgl1, macrophage galactose-type C-type lectin 1; Arg1,arginase 1; Il-10, interleukin 10; Il-4, interleukin 4. Data are mean ± SD (n = 9–10); *p<0.05 (Student’s t-test). (C) Representative hematoxylin and eosin (H&E) staining (left) and immunohistochemical anti-MAC-2 staining (right; brown color) in BAT from PBS and DT mice. Scale bar 100 μm. Quantification of MAC-2 positive area (panel below MAC-2 staining) as a percentage of total area.</p