The effects of anesthetic agents, commonly used in animal models, on blood glucose levels in fed and fasted rats were investigated. In fed Sprague-Dawley rats, ketamine (100 mg/kg)/xylazine (10 mg/kg) (KX) produced acute hyperglycemia (blood glucose 178.4 +/- 8.0 mg/dl) within 20 min. The baseline blood glucose levels (104.8 +/- 5.7 mg/dl) reached maximum levels (291.7 +/- 23.8 mg/dl) at 120 min. Ketamine alone did not elevate glucose levels in fed rats. Isoflurane also produced acute hyperglycemia similar to KX. Administration of pentobarbital sodium did not produce hyperglycemia in fed rats. In contrast, none of these anesthetic agents produced hyperglycemia in fasted rats. The acute hyperglycemic effect of KX in fed rats was associated with decreased plasma levels of insulin, adrenocorticotropic hormone (ACTH), and corticosterone and increased levels of glucagon and growth hormone (GH). The acute hyperglycemic response to KX was dose-dependently inhibited by the specific alpha2-adrenergic receptor antagonist yohimbine (1-4 mg/kg). KX-induced changes of glucoregulatory hormone levels such as insulin, GH, ACTH, and corticosterone were significantly altered by yohimbine, whereas the glucagon levels remained unaffected. In conclusion, the present study indicates that both KX and isoflurane produce acute hyperglycemia in fed rats. The effect of KX is mediated by modulation of the glucoregulatory hormones through stimulation of alpha2-adrenergic receptors. Pentobarbital sodium did not produce hyperglycemia in either fed or fasted rats. Based on these findings, it is suggested that caution needs to be taken when selecting anesthetic agents, and fed or fasted state of animals in studies of diabetic disease or other models where glucose and/or glucoregulatory hormone levels may influence outcome and thus interpretation. However, fed animals are of value when exploring the hyperglycemic response to anesthetic agents.

Abstract. Connective tissue growth factor (CTGF) is a peptide secreted by cultured endothelial cells and fibroblasts when stimulated by transforming growth factor-β (TGF-β), and is overexpressed during fibrotic processes in coronary arteries and in skin. To determine whether CTGF is implicated in the pathogenesis of diabetic glomerulosclerosis, cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli were examined from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to recombinant human CTGF significantly increased fibronectin and collagen type I production. Furthermore, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36 to 38 kD) into the media. However, sodium heparin treatment resulted in a greater than fourfold increase in media-associated CTGF, suggesting that the majority of CTGF produced was cell- or matrix-bound. Exposure of MC to TGF-β, increased glucose concentrations, or cyclic mechanical strain, all causal factors in diabetic glomerulosclerosis, markedly induced the expression of CTGF transcripts, while recombinant human CTGF was able to autoinduce its own expression. TGF-β and high glucose, but not mechanical strain, stimulated the concomitant secretion of CTGF protein, the former also inducing abundant quantities of a small molecular weight form of CTGF (18 kD) containing the heparin-binding domain. The induction of CTGF protein by a high glucose concentration was mediated by TGF-β, since a TGF-β-neutralizing antibody blocked this stimulation. In vivo studies using quantitative reverse transcription-PCR demonstrated that although CTGF transcripts were low in the glomeruli of control mice, expression was increased 28-fold after approximately 3.5 mo of diabetes. This change occurred early in the course of diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (twofold) observed in whole kidney cortices indicated that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis, acting downstream of TGF-β.

The present studies identified a novel DPP‐4 resistant GLP‐1 analogue info:ddbj-emblgenbank/LY548806 and characterized its pharmacological activity in a rat model of acute hyperglycemia. info:ddbj-emblgenbank/LY548806 was more than 20‐fold less susceptible to DPP‐4 cleavage and exhibited a 50‐fold greater physical stability over native GLP‐1, and was a full agonist with an EC50 of 10.6 ± 1.6 pM in vitro. In an acute hyperglycemia model, continuous infusion of info:ddbj-emblgenbank/LY548806 (0.1–10 μg/kg/hr) or insulin (0.01 U–0.1U/kg/hr) via the right jugular vein resulted in dose‐dependent reduction of glucose levels with estimated ED50 values of 0.32 ± 0.11 ug and 0.08 ± 0.00 U respectively. In contrast to insulin, info:ddbj-emblgenbank/LY548806 did not produce hypoglycemia at higher dose levels. Continuous infusion of info:ddbj-emblgenbank/LY548806 (10 μg/kg/hr) or insulin (0.1 U/kg/hr) did not affect the plasma levels of the endogenous glucoregulatory hormones corticosterone, growth hormone, catecholamines, or insulin, indicating an insulin‐independent effect of info:ddbj-emblgenbank/LY548806 in this model. info:ddbj-emblgenbank/LY548806 significantly lowered the plasma levels of free fatty acids (FFA) and glucagon, whereas insulin only reduced FFA levels. In conclusion, we have identified a stable GLP‐1 analogue with improved solubility which effectively ameliorates acute hyperglycemia without the risk of hypoglycemia and may represent a novel therapeutic strategy for acute critical illness associated with acute hyperglycemia.

Assessment of pharmacodynamic (PD) effect of novel therapeutic compounds in human tissues is used to demonstrate target engagement and to rationalize dosing strategy decisions for emerging targeted therapeutics in clinical trials. Often, human skin biopsies, a readily accessible surrogate tissue, are used for these purposes. Aberrant signaling in the Notch cell surface receptor and the PI3K signal transduction pathways are increasingly implicated in human cancers. In order to test the utility of immunohistochemical (IHC) PD biomarker assays developed in our laboratory for Notch and PI3K inhibitor development programs, we developed ex vivo PD models using respective inhibitors on human skin biopsy explants. Fresh human skin samples were obtained in the Operating-Room from reduction mammoplasty specimens, serially sliced (1-2 mm) and incubated at 37°C in Williams E. media containing 1 µM Notch (gamma-secretase) inhibitor (15 h); and 50 µM PI3K inhibitor (LY294002, 4 h). Specimens were fixed in 10% neutral buffered formalin for 24 hours, and processed into FFPE blocks. Sections were stained for Notch1 intracellular domain (N1ICD, rabbit monoclonal, D3B8) using DAB and for phospho-S6 (S240/S244, rabbit polyclonal) using an alkaline phosphatase-based detection system. N1ICD and phospho-S6 immunoreactivity in cells of the epidermis was analyzed using positive pixel counting. Viability of epidermal cells throughout ex vivo incubation was verified by Ki67 and histomorphologic assessments of the epidermis. Nuclear N1ICD immunoreactivity, a marker of Notch pathway activation, was decreased by 76% when treated with gamma-secretase inhibitor. Similarly, treatment with PI3K inhibitor decreased cytoplasmic phospho-S6 immunoreactivity by 98%. These ex vivo models of human skin provide scientific evidence to support: 1) The N1ICD and phospho-S6 IHC assays developed in our laboratory can demonstrate ex vivo inhibition of these two pathways in human skin; and 2) Feasibility to translate human skin explant findings to patient skin samples from clinical trials. Our explant model systems are closer to real life PD assessments on patient skin samples from clinical trials and can further substantiate the PD data generated in animal models. In some cases, the ex vivo model could replace animal models. Citation Format: Timothy R. Holzer, Leslie A. O'Neill, Angie D. Fulford, Janet M. Grondin, Bradley L. Ackermann, Robert J. Konrad, Kelly M. Credille, Aejaz Nasir. Demonstration of pharmacodynamic effects of Notch and PI3Kinase inhibitors using robust immunohistochemical assays on human skin explant models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2014-4567