Effect of adding a phytogenic-rich herbal mixture to diet on the expression pattern of some insulin hormone metabolism-related candidate genes of heat-stressed fattening Afshari-Shal lambs

Introduction: Heat stress can compromise sheep's productive performance and growing lambs are considered to be one of the most susceptible groups to heat stress. Exposure to heat stress increases inflammatory cytokines secretion and impairs insulin signaling in muscle which may underlie poor growth responses. Therefore, enhancing insulin sensitivity through the addition of insulin-sensitizing dietary additives may improve animal performance during heat stress. Phytogenics, compounds with antioxidant, antimicrobial, and metabolic enhancing effects, have been used in animal nutrition to enhance performance and prevent diseases in stress-related conditions. It has been reported that supplementing a phytogenic-rich herbal mixture containing clove, rosemary, cinnamon, and turmeric improved the antioxidant status and enhanced insulin sensitivity of transition dairy cows. It has been indicated that the duodenal infusion of quercetin increased insulin secretion and insulin sensitivity. Possible mechanisms responsible for the insulin-sensitizing activity of herbal plants include increasing insulin receptors or enhancing receptor sensitivity to insulin, increasing PPARy activity, increasing glycogen synthase activity, modulating inflammatory cytokine expression, stimulating fatty acid oxidation as well as increasing antioxidant status. The objective of this study was to investigate the effects of a phytogenic-rich herbal mixture (PRHM) supplementation on gene expression of insulin-related genes in the muscle of feedlot lambs experiencing severe heat stress conditions. Materials and methods: Eighteen 11-12-month-old growing male Afshari×Chaal lambs (41.2 ± 3.04 kg) were housed in individual stalls (1.2 × 1 m, with individual feed troughs) on wooden slatted flooring, located within an indoor animal facility with a natural and artificial lighting system for 16 h/d. Lambs were randomly allocated to one of three experimental diets for a 48-day feeding period. The treatments included control diet without PRHM (0%PRHM), diet supplemented with 1% PRHM (1%PRHM), and diet supplemented with 2% PRHM (2%PRHM). The PRHM was added to the basal diet and mixed thoroughly. Diets formulated to meet nutritional requirements for maintenance and growth and offered twice daily at 0700 and 1700 h. The lambs had free access to feed and water during the experiment. The main ingredients of the basal diet were alfalfa hay, ground corn, wheat bran, beet pulp, soybean meal, and soy oil. The herbal mixture consisted of 50% rosemary leaves (Rosemarinus offıcinalis), 20% cinnamon barks (Cinnamomum zeylanicum), 20% turmeric roots (Curcuma longa), and 10% clove buds (Eugenia caryophyllata Thunb.). The total phenolic content of the PRHM was 89.6 g tannic acid equivalent per kg. The daily climate data, such as daily mean temperature and relative humidity, were recorded using a digital thermo-hygrometer (Testo 608-H1, Germany). At the end of the study, all lambs were transported to a local abattoir where they were slaughtered after 12-h feed withdrawal according to Halal method. Post-slaughter, longissimus lumborum muscle samples between the 12th and the 13th rib of the right side of carcass were collected as quickly as possible (i.e., within 3–5 min). For expression analysis of Insulin receptor (INSR), Insulin receptor substrate 1 (IRS1), glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) genes, total RNA was isolated from the sheep muscle tissue using the SinaPure RNA according to the manufacturer’s instructions. The quantity and integrity of isolated RNA were determined for each sample by using both NanoDrop (260/280 ratio) and 1% agarose gel electrophoresis. cDNA was synthesized with RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. Forward and reverse primers were designed for five genes and GAPDH as reference gene by Oligo 7. The real-time PCR was done in 25 µL containing 1x BIORON GreenHot Master Mix (BIORON, Germany), 1 µL cDNA, 0.3 µM of each forward and reverse primers and nuclease-free water to reach final volume. Real-time PCR was performed on Bio-Rad CFX96 as follows; Initial denaturation 2 min at 94 °C, followed by 39 cycles of denaturation 20 sec at 94 °C, annealing 25 sec at annealing temperature, extension 20 sec at 72 °C following by melting curve analysis from 65 °C to 95 °C with 0.5 °C increase per each step. Relative gene expression was determined based on Livak method (2-ΔΔCT). All data were analyzed using the MIXED procedure of SAS. Model included the effect of the treatment as the fixed effect and lambs within treatments as a random effect. The significance level was declared at P<0.05. Results and discussion: The results showed that the gene expressions of INSR (P<0.01) and GLUT4 (P<0.01) in muscle tissue were linearly upregulated in lambs supplemented with increasing levels of herbal mixture. A quadratic effect of herbal mixture supplementation was found on gene expression of IRS1 such that lambs fed a 2% mixture had the highest gene expression. Moreover, both supplementation levels of 1 and 2% mixture equally increased GLUT1 gene expression (P<0.01) compared to control lambs. In contrast to the other genes, PPARγ gene expression was quadratically (P<0.01) downregulated by mixture supplementation such that the lowest gene expression was observed in lambs receiving a 2% mixture. Although there is some evidence suggesting that PRHM supplementation has modulatory effects on insulin resistance in transition dairy cows, to our knowledge, there is currently no published data investigating the effects of PRHM supplementation on mRNA expression of genes related to insulin and glucose metabolism in lambs’ skeletal muscle. Our results may suggest that PRHM supplementation enhanced insulin sensitivity and glucose uptake in the skeletal muscle of heat-stressed lambs. In agreement with our results, it has been found that cinnamaldehyde promotes gene expression of IRS-1 and GLUT4 genes in muscle cells, which the latter gene is affected by activating the peroxisome proliferator-activated receptor gamma coactivator 1-alpha. In addition, greater GLUT1 gene expression might be indicative of improved insulin-independent glucose uptake, besides enhanced insulin-dependent glucose transporter, GLUT4 gene. Conclusions: The results showed that the mixture of medicinal plants including cinnamon, turmeric, rosemary, and clove affected the expression of genes related to insulin metabolism and increased the efficiency of glucose utilization.