Glucagon receptor activation contributes to the development of kidney injury.

The underlying causes of diabetic kidney disease are still largely unknown. New insights into the contributing causes of diabetic nephropathy are important in order to prevent this complication. Hyperglycemia and hypertension are some of the risk factors for diabetic nephropathy. However, the incidence of diabetic nephropathy is increasing despite efforts to normalize blood-glucose levels and blood pressure. Therefore, other factors should be investigated as causes of diabetic nephropathy. We investigated whether long-term increased plasma levels of glucagon contribute to the development of pathophysiological changes in kidney function as seen in patients with diabetic nephropathy. Using mouse models of chronic activation and inactivation of glucagon receptor signaling, we investigated whether glucagon is involved in changes in renal function, renal structural and transcriptional changes. We found several histopathological changes in the kidney, such as thickening of the parietal layer of Bowman's capsule, glomerular mesangial cell expansion, and significant albuminuria in the mice with activated glucagon receptor signaling. Opposite effects on mesangial area expansion and the development of albuminuria were demonstrated in mice with glucagon receptor inactivation. RNA sequencing data revealed that transcription of genes related to fatty acid metabolism, podocytes, Na+/K+-ATPase, and sodium/glucose transport were significantly changed in mice with activated glucagon receptor signaling. These data implicate that the glucagon receptor signaling is involved in the development of kidney injury, as seen in type 2 diabetes and that glucagon receptor is a potential therapeutic target in the treatment of diabetes.

In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation.

BACKGROUND: Insulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent. METHODS: Insulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements. RESULTS: We quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided. CONCLUSION: We present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.

L-valine is a powerful stimulator of GLP-1 secretion in rodents and stimulates secretion through ATP-sensitive potassium channels and voltage-gated calcium channels.

BACKGROUND: We previously reported that, among all the naturally occurring amino acids, L-valine is the most powerful luminal stimulator of glucagon-like peptide 1 (GLP-1) release from the upper part of the rat small intestine. This makes L-valine an interesting target for nutritional-based modulation of GLP-1 secretion. However, the molecular mechanism of L-valine-induced secretion remains unknown. METHODS: We aimed to investigate the effect of orally given L-valine in mice and to identify the molecular details of L-valine stimulated GLP-1 release using the isolated perfused rat small intestine and GLUTag cells. In addition, the effect of L-valine on hormone secretion from the distal intestine was investigated using a perfused rat colon. RESULTS: Orally given L-valine (1 g/kg) increased plasma levels of active GLP-1 comparably to orally given glucose (2 g/kg) in male mice, supporting that L-valine is a powerful stimulator of GLP-1 release in vivo (P > 0.05). Luminal L-valine (50 mM) strongly stimulated GLP-1 release from the perfused rat small intestine (P < 0.0001), and inhibition of voltage-gated Ca2+-channels with nifedipine (10 µM) inhibited the GLP-1 response (P < 0.01). Depletion of luminal Na+ did not affect L-valine-induced GLP-1 secretion (P > 0.05), suggesting that co-transport of L-valine and Na+ is not important for the depolarization necessary to activate the voltage-gated Ca2+-channels. Administration of the KATP-channel opener diazoxide (250 µM) completely blocked the L-valine induced GLP-1 response (P < 0.05), suggesting that L-valine induced depolarization arises from metabolism and opening of KATP-channels. Similar to the perfused rat small intestine, L-valine tended to stimulate peptide tyrosine-tyrosine (PYY) and GLP-1 release from the perfused rat colon. CONCLUSIONS: L-valine is a powerful stimulator of GLP-1 release in rodents. We propose that intracellular metabolism of L-valine leading to closure of KATP-channels and opening of voltage-gated Ca2+-channels are involved in L-valine induced GLP-1 secretion.

Evaluation of commercially available glucagon receptor antibodies and glucagon receptor expression.

Glucagon is a major regulator of metabolism and drugs targeting the glucagon receptor (GCGR) are being developed. Insight into tissue and cell-specific expression of the GCGR is important to understand the biology of glucagon and to differentiate between direct and indirect actions of glucagon. However, it has been challenging to localize the GCGR in tissue due to low expression levels and lack of specific methods. Immunohistochemistry has frequently been used for GCGR localization, but antibodies targeting G-protein-coupled-receptors may be inaccurate. We evaluated all currently commercially available GCGR antibodies. The antibody, ab75240 (Antibody no. 11) was found to perform best among the twelve antibodies tested and using this antibody we found expression of the GCGR in the kidney, liver, preadipocytes, pancreas, and heart. Three antibody-independent approaches all confirmed the presence of the GCGR within the pancreas, liver and the kidneys. GCGR expression should be evaluated by both antibody and antibody-independent approaches.