The Effects of Tetrapeptides Designed to Fit the Androgen Binding Site of ZIP9 on Myogenic and Osteogenic Cells

ZIP9 is a recently identified membrane-bound androgen receptor of physiological significance that may mediate certain physiological responses to androgens. Using in silico methods, six tetrapeptides with the best docking properties at the testosterone binding site of ZIP9 were synthesized and further investigated. All tetrapeptides displaced T-BSA-FITC, a membrane-impermeable testosterone analog, from the surface of mouse myogenic L6 cells that express ZIP9 but not the classical androgen receptor (AR). Silencing the expression of ZIP9 with siRNA prevented this labeling. All tetrapeptides were found to be pro-androgenic; in L6 cells they stimulated the expression of myogenin, triggered activation of focal adhesion kinase, and prompted the fusion of L6 myocytes to syncytial myotubes. In human osteoblastic SAOS-2 cells that express AR and ZIP9, they reduced the expression of alkaline phosphatase and stimulated mineralization. These latter effects were prevented by silencing ZIP9 expression, indicating that the osteoblast/osteocyte conversion is exclusively mediated through ZIP9. Our results demonstrate that the synthetic tetrapeptides, by acting as ZIP9-specific androgens, have the potential to replace testosterone or testosterone analogs in the treatment of bone- or muscle-related disorders by circumventing the undesirable effects mediated through the classical AR.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines

Background: Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α–amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

In vitro evaluation of the anti-diabetic potential of Helichrysum petiolare Hilliard & B.L. Burtt using HepG2 (C3A) and L6 cell lines

Background: Helichrysum petiolare Hilliard & B.L. Burtt has been listed in a survey of plants used in traditional medicine for the treatment of type 2 diabetes in the Eastern Cape of South Africa. In this study, the antidiabetic potentials of ethanol, cold aqueous (CAQ) and boiled aqueous (BAQ) extracts of H. petiolare were investigated. Methods: The cytotoxic and glucose utilization effects of the extracts were evaluated using L6 myocytes and HepG2 (C3A) hepatocytes. α-amylase, α-glucosidase and lipase inhibition assays were also carried out. Results: The ethanol extract showed significant cytotoxic effects in the treated cells. Both BAQ and CAQ extracts significantly increased glucose uptake in L6 and C3A cell lines. The CAQ extract enhanced glucose uptake more in the L6 myocytes than in the C3A cell-lines hepatocytes. The BAQ extract showed higher levels of inhibition on α–amylase and α-glucosidase than CAQ. The activities were not significantly different from acarbose. However, BAQ showed lower lipase inhibition than acarbose (p<0.05). Conclusions: The BAQ and CAQ extracts of H. petiolare may, therefore, contain pharmacologically active and relatively non-toxic hypoglycaemic chemicals, which may be effective substitutes in the treatment of diabetes mellitus.

Effects of Retinoic Acid and Insulin on Glycogen Content and Expression Levels of Glycogenic Proteins in Differentiating L6 Myocytes

Objectives The skeletal muscle is critical for the control of glucose homeostasis partially through glycogenesis. The effects of retinoic acid (RA) on glycogen content and expressions of key glycogenic proteins without or with insulin in L6 rat myocytes deserve to be studied. Methods L6 myocytes at confluence were induced to differentiation using DMEM with 2% horse serum. Cells were incubated in medium with vehicle control or 1 μM RA without or with 10 nM insulin for 2, 4, and 6 days. Glycogen was extracted and analyzed using α-amyloglucosidase and released glucose assay. Proteins in cell lysates from parallel groups were extracted and subjected to analysis using western blotting. The expression levels of glycogen synthase (GS), phospho-glycogen synthase (Ser 641) (p-GS), glycogen synthase kinase 3-β(GSK3β), and phospho-glycogen synthase kinase 3-β (p-GSK3β) were determined using their specific antibodies, and then quantified with ImageJ software. Results The RA + insulin group had higher glycogen content than the control group on days 2 to 6. A synergistic effect of RA and insulin were observed on day 2. GS expression was synergistically induced in the RA + insulin group on each day. The total p-GS normalized to β-actin in the RA + insulin group was higher than that in the control on day 2. However, the ratio of p-GS/GS in the RA + insulin group was lower than that in the control on day 2 and in RA group on day 6, respectively. GSK3β expression levels in insulin and RA + insulin groups were higher than that in the control on day two. The total p-GSK3β normalized to β-actin showed an increase in the insulin group compared to the RA group on day 6. The p-GSK3β/GSK3β ratio in the insulin group was higher than that in RA and RA + insulin groups on day 6. Conclusions Treatments with RA and insulin increase the glycogen content in L6 cells via upregulation of glycogenesis. A synergy of RA and insulin can be seen. The increases of total GS and decreases of p-GS/GS ratio contribute to the elevation of GS activity. RA likely exerts its glycogenic effects through changes of the total GS level and phosphorylation by GSK3β. Funding Sources Internal fund at the Univenrsity of Tennessee.

Dietary n-3 vs n-6 PUFA Differentially Modulate Macrophage-Myocyte Inflammatory Cross-Talk

Objectives Skeletal muscle is the primary site for insulin-stimulated glucose uptake. In obesity, increased circulating inflammatory cytokines interfere with skeletal muscle insulin signaling, leading to local and whole-body insulin resistance (IR). Moreover, obese skeletal muscle is characterized by accumulation of infiltrated M1 macrophages and ensuing macrophage-myocyte paracrine interactions (cross-talk) contribute to local inflammation and IR. Such macrophage-myocyte inflammatory cross-talk provides a potential intervention target for anti-inflammatory nutrients, including dietary long-chain n-3 polyunsaturated fatty acids (PUFA). Methods Using a co-culture model designed to mimic the degree of CD11b+ cell accumulation in obese skeletal muscle (40% of immune cells), differentiated L6 myocytes were co-cultured with purified splenic CD11b+ cells from male Sprague Dawley rats (7-wk old) consuming one of three isocaloric diets: i) high-fat (HF; 54% kcal lard, 6% kcal soybean oil), ii) high-fat with n-3 PUFA (HFn-3; 39% kcal lard + 15% kcal menhaden oil + 6% kcal soybean oil) or iii) high-fat with n-6 PUFA (HFn-6; 45% kcal lard + 15% kcal soybean oil) for 2, 8 or 12-wk (n = 8–12/diet). Co-cultures were stimulated for 24 h with lipopolysaccharide (LPS, 10 ng/mL) to mimic in vivo obese endotoxin levels. CD11b+ cells were also cultured alone for 24 h in conditioned media collected from L6 myocytes stimulated with LPS for 24 h (LCM). Results In co-cultures, HFn-6 increased mRNA expression of inflammatory markers compared to HF and HFn-3 at 8- (iNos; P ≤ 0.05) and 12-wk (Tnf-α, Il-6, Il-1β; P ≤ 0.05). Similarly, at 8-wk CD11b+ cells from HFn-6 rats that were treated with LCM, had increased mRNA expression of inflammatory cytokines (Tnf-α, Il-1β) and M1 polarization markers (iNos, Cd86) compared to both HF and HFn-3, and the same effects were seen with Il-6 and Il-1β at 12-wk (P ≤ 0.05). Lastly, HFn-3 reduced mRNA expression of Tnf-α compared to HF at 12-wk (P ≤ 0.05). Conclusions Together, these data suggest that n-6 PUFA support macrophage-myocyte inflammatory cross-talk, in part by promoting M1 macrophage polarization. Further, these data provide mechanistic insight into the benefits of n-3 vs n-6 PUFA inclusion in a high-fat diet in mitigating skeletal muscle inflammation in obesity. Funding Sources Natural Sciences and Engineering Research Council (NSERC) of Canada.