Gene Expression Signature in Adipose Tissue of Acromegaly Patients.

To study the effect of chronic excess growth hormone on adipose tissue, we performed RNA sequencing in adipose tissue biopsies from patients with acromegaly (n = 7) or non-functioning pituitary adenomas (n = 11). The patients underwent clinical and metabolic profiling including assessment of HOMA-IR. Explants of adipose tissue were assayed ex vivo for lipolysis and ceramide levels. Patients with acromegaly had higher glucose, higher insulin levels and higher HOMA-IR score. We observed several previously reported transcriptional changes (IGF1, IGFBP3, CISH, SOCS2) that are known to be induced by GH/IGF-1 in liver but are also induced in adipose tissue. We also identified several novel transcriptional changes, some of which may be important for GH/IGF responses (PTPN3 and PTPN4) and the effects of acromegaly on growth and proliferation. Several differentially expressed transcripts may be important in GH/IGF-1-induced metabolic changes. Specifically, induction of LPL, ABHD5, and NRIP1 can contribute to enhanced lipolysis and may explain the elevated adipose tissue lipolysis in acromegalic patients. Higher expression of TCF7L2 and the fatty acid desaturases FADS1, FADS2 and SCD could contribute to insulin resistance. Ceramides were not different between the two groups. In summary, we have identified the acromegaly gene expression signature in human adipose tissue. The significance of altered expression of specific transcripts will enhance our understanding of the metabolic and proliferative changes associated with acromegaly

Clinical characteristics.

<p>Data represents mean +/- standard error.</p><p>Clinical characteristics.</p

Donor-Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light.

Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry; however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling remains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship that enables optical triggering with visible light whilst simultaneously attenuating the probe's photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of the probe and short irradiation times (10-60 min) with violet light enabled efficient reactivity toward surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescence quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 440 nm LED irradiation. Peptide-level enrichment from live cell labeling experiments identified 290 Trp modifications, with 82% selectivity for Trp modification over other π-amino acids, demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells

Acromegalic patients have reduced insulin sensitivity and trend for higher lipolytic activity than their controls.

<p>A) Fasting blood glucose levels. B) Fasting insulin levels. C) HOMA-IR score from Control or Acromegaly subjects. D) <i>ex vivo</i> lipolysis as measured by glycerol release from excised white adipose tissue from control or acromegaly patients left untreated (Basal) or after stimulation with 30 nM isoproterenol (Iso). Data is presented as mean +/- standard error of the mean. Asterisk indicates q<0.05.</p

Differential expression of genes in white adipose tissue from subjects with acromegaly compared to controls.

<p>A) Heatmap of the differentially expressed genes in white adipose tissue. Individual values are colored as the log fold change for a particular gene in a particular subject compared to the average expression of that gene across all cohorts, with brown indicating less expression and green indicating more expression (designated in the key as Row Z-score). The bar across the top indicates the subject’s diagnosis, red for acromegaly and blue for controls. B) Scatterplot showing the log2 fold change for genes which had a statistically significant difference (q<0.05) between acromegaly and control subjects. Each dot represents the log2 fold change for acromegaly for a gene in the under 60 and 60 or over cohorts. The solid line represents a slope of 1, which would imply no difference in fold change between age groups. The red line is a best fit line with a lower slope, showing that on average the fold change for older patients is smaller than the fold change for the under-60 patients.</p

Expression changes of selected genes, potentially responsible for lipolytic or insulin sensitivity alterations in acromegaly patients.

<p>mRNA Expression profile of genes potentially involved in lipid catabolism (A), regulation of lipolysis (B), fatty acid synthesis (C), fatty acid desaturation (D), <i>TCF7L2</i> (E), and glucocorticoid signaling (F). Asterisks indicate q<0.05. Data indicates mean +/- standard error of the mean.</p

GH targets are differentially expressed in acromegaly subjects.

<p>A) mRNA Expression of A) <i>IGF1</i> and B) <i>IGFBP3</i> transcript levels in adipose tissue from control and acromegalic patients (C) Comparason between <i>IGF1</i> mRNA and IGF-1 serum levels in patients with acromegaly (D and E) Expression of mRNA for suppressors of growth hormone signaling (F) and Expression of tyrosine phosphatases associated with growth hormone signaling. Asterisks indicate q<0.05 for the separated under 60 and 60 or over cohorts for panels A, B, D and E and for the age adjusted combined analysis for panel F. Barplots are presented as mean +/- standard error of the mean.</p