Short communication: Protein kinase C regulates glucose uptake and mRNA expression of glucose transporter (GLUT) 1 and GLUT8 in lactating bovine mammary epithelial cells

The aim of this study was to determine the role of protein kinase C (PKC) in regulating glucose uptake in lactating bovine mammary epithelial cells (BMEC). The BMEC were cultured and treated with different concentrations of phorbol 12-myristate 13-acetate (PMA;0, 10, 25, 50, 100, and 200 ng/mL), the classic activator of PKC, for 48 h. Compared with the cells with no PMA treatment, 50 and 100 ng of PMA/mL significantly stimulated the glucose uptake of the BMEC, whereas the glucose uptake by the cells treated with the lowest and the highest amounts of PMA (25 and 200 ng/mL, respectively) did not show a significant difference. Consistently, the mRNA expression of glucose transporter (GLUT) 1 and 8 showed a similar pattern of increase under the treatments of PMA. Furthermore, when the cells were pretreated with GF1090203X (0, 0.25, 0.5, 1, and 2 μM), an inhibitor of PKC, for 30 min before exposed to PMA (50 ng/mL), the PMA-induced glucose uptake and GLUT1 and GLUT8 expression were decreased by GF1090203X in a dose-dependent manner. These results demonstrate that PKC is involved in the regulation of glucose uptake by BMEC, and this function may work, at least partly, through upregulating the expression of GLUT1 and GLUT8.     

Effects of estradiol on bovine thecal cell function in vitro: dependence on insulin and gonadotropins

The objective of this study was to evaluate the influence of estradiol (E2) on proliferation and steroid production by thecal cells obtained from large (≥8 mm) follicles of cattle. Five experiments evaluated the effect of various doses of E2 during a 2-d exposure in serum-free medium on hormone-induced steroidogenesis and cell proliferation. In LH-treated thecal cells of experiment 1, 300 ng/mL of E2 decreased progesterone production by 30% and increased androstenedione production to 5.8-fold of controls. In the absence of LH, both 3 and 300 ng/mL of E2 increased progesterone production. In experiment 2, in the presence of insulin and LH, 3, 30, and 300 ng/mL of E2 decreased progesterone production (by 17 to 36%), whereas 3 ng/mL of E2 decreased and 300 ng/mL of E2 increased androstenedione production. Doses of LH (3 to 30 ng/mL) tested in experiment 3 increased (to as much as 3.7-fold) progesterone production by thecal cells and E2 attenuated this stimulatory effect by 40%. In contrast, E2 amplified the stimulatory effect of LH on androstenedione production in experiment 3. In experiment 4, E2 (300 ng/mL) decreased IGF-I- and insulin-induced thecal cell progesterone production by 70 to 77%, whereas E2 increased basal, IGF-I, and insulin-induced androstenedione production. In experiment 5, in the presence of insulin, 10 to 1000 ng/mL of E2 had no effect on [¹²⁵I]-IGF-I binding to thecal cells, whereas 10 and 100 ng/mL of E2 increased and 1000 ng/mL of E2 decreased progesterone production by thecal cells. Estradiol had no consistent effect on thecal cell numbers among the 5 experiments. These results support the hypothesis that E2 may act as a paracrine factor to directly regulate hormone-induced steroid production by thecal cells without affecting cell numbers or numbers of insulin-like growth factor type I receptors.     

Short communication: Glucose infusion into early postpartum cows defines an upper physiological set point for blood glucose and causes rapid and reversible changes in blood hormones and metabolites

Low blood glucose concentrations after calving are associated with infertility in postpartum dairy cows perhaps because glucose is a master regulator of hormones and metabolites that control reproductive processes. The hypothesis was that low blood glucose postpartum is caused by inadequate glucose entry rate relative to whole-body demand as opposed to the alternative possibility that postpartum cows have a lower regulatory set point for blood glucose. Eight early postpartum (10 to 25 d) dairy cows (5 Holstein and 3 Guernsey) were jugular catheterized. During the first 24 h, cows were infused with physiological saline at 83.3 mL/h. After 24 h, the infusion solution was switched to 50% dextrose that was infused at a rate of 41.7 mL/h (total daily glucose dose = 500 g). On d 3 and d 4, the rate of glucose infusion was increased to 83.3 mL/h (daily dose = 1,000 g) and 125 mL/h (daily dose = 1,500 g), respectively. On d 5, physiological saline was infused at 83.3 mL/h. Blood was sampled hourly through a second jugular catheter (contralateral side) and analyzed for glucose, nonesterified fatty acids, β-hydroxybutyrate, insulin-like growth factor 1, and insulin. Blood glucose concentrations on d 1 (saline infusion) averaged 53.4 ± 1.7 mg/dL. Blood glucose concentrations increased on d 2 when cows were infused with 500 g/d and increased further on d 3 when cows were infused with 1,000 g of glucose/d. Increasing the infusion rate to 1,500 g/d on d 4 did not cause a further increase in blood glucose concentrations. Based on a segmented regression analysis, the upper physiological set point for blood glucose was 72.1 mg/dL. Both insulin and insulin-like growth factor 1 concentrations increased in response to glucose infusion and decreased when cows were infused with saline on d 5. Serum nonesterified fatty acids and β-hydroxybutyrate concentrations decreased in response to glucose infusion and rebounded upward on d 5 (saline infusion). In conclusion, early postpartum cows had circulating blood glucose concentrations that were well below the upper set point defined in this study (72.1 mg/dL). Infusing approximately 1,000 g of glucose daily increased blood glucose to the physiological set point and rapidly changed the hormonal and metabolic profile that typifies postpartum cows. The inability of the early postpartum cow to achieve an adequate entry rate for glucose relative to whole-body demand is a possible mechanism that links postpartum physiology and nutrition to reproduction in dairy cows.     

Adipose tissue insulin receptor and glucose transporter 4 expression,and blood glucose and insulin responses during glucose tolerance tests in transition Holstein cows with different body condition

Glucose uptake in tissues is mediated by insulin receptor (INSR) and glucose transporter 4 (GLUT4). The aim of this study was to examine the effect of body condition during the dry period on adipose tissue mRNA and protein expression of INSR and GLUT4, and on the dynamics of glucose and insulin following the i.v. glucose tolerance test in Holstein cows 21 d before (d ?21) and after (d 21) calving. Cows were grouped as body condition score (BCS) ≤3.0 (thin, T; n = 14), BCS = 3.25 to 3.5 (optimal, O; n = 14), and BCS ≥3.75 (overconditioned, OC; n = 14). Blood was analyzed for glucose, insulin, fatty acids, and β-hydroxybutyrate concentrations. Adipose tissue was analyzed for INSR and GLUT4 mRNA and protein concentrations. During the glucose tolerance test 0.15 g/kg of body weight glucose was infused; blood was collected at ?5, 5, 10, 20, 30, 40, 50, and 60 min, and analyzed for glucose and insulin. On d ?21 the area under the curve (AUC) of glucose was smallest in group T (1,512 ± 33.9 mg/dL × min) and largest in group OC (1,783 ± 33.9 mg/dL × min), and different between all groups. Basal insulin on d ?21 was lowest in group T (13.9 ± 2.32 µU/mL), which was different from group OC (24.9 ± 2.32 µU/mL. On d ?21 the smallest AUC 5–60 of insulin in group T (5,308 ± 1,214 µU/mL × min) differed from the largest AUC in group OC (10,867 ± 1,215 µU/mL × min). Time to reach basal concentration of insulin in group OC (113 ± 14.1 min) was longer compared with group T (45 ± 14.1). The INSR mRNA abundance on d 21 was higher compared with d ?21 in groups T (d ?21: 3.3 ± 0.44; d 21: 5.9 ± 0.44) and O (d ?21: 3.7 ± 0.45; d 21: 4.7 ± 0.45). The extent of INSR protein expression on d ?21 was highest in group T (7.3 ± 0.74 ng/mL), differing from group O (4.6 ± 0.73 ng/mL), which had the lowest expression. The amount of GLUT4 protein on d ?21 was lowest in group OC (1.2 ± 0.14 ng/mL), different from group O (1.8 ± 0.14 ng/mL), which had the highest amount, and from group T (1.5 ± 0.14 ng/mL). From d ?21 to 21, a decrease occurred in the GLUT4 protein levels in both groups T (d ?21: 1.5 ± 0.14 ng/mL; d 21: 0.8 ± 0.14 ng/mL) and O (d ?21: 1.8 ± 0.14 ng/mL; d 21: 0.8 ± 0.14 ng/mL). These results demonstrate that in obese cows adipose tissue insulin resistance develops prepartum and is related to reduced GLUT4 protein synthesis. Regarding glucose metabolism, body condition did not affect adipose tissue insulin resistance postpartum.     

Coordination of mammary metabolism and blood flow after refeeding in rats

The production of milk is closely linked to nutritional state in many mammalian species, but the mechanisms by which changes in nutritional state are signaled to the mammary glands are poorly understood. Simultaneous measurements of mammary blood flow and glucose arterio-venous difference were made across the inguinal mammary glands of anesthetized, lactating rats. Blood flow to the mammary glands of previously fed rats was 0.48 mL/min per gram of mammary tissue. Glucose supply was 1.7 μmol/min per gram and 28% was extracted by the mammary glands. After food deprivation for 18 h, mammary blood flow decreased 48%, glucose arterio-venous difference decreased 72%, and hematocrit increased 7%, resulting in a 60% decrease in glucose supply and an 88% decrease in glucose uptake. After 1 h of refeeding, glucose supply had returned to a similar level to that of normally fed animals, but glucose uptake was 60% higher than in the normally fed state. Mammary glucose uptake was not closely linked to either blood flow or glucose supply, suggesting that substrate supply was not the primary determinant of mammary metabolism. Denervation experiments showed that the mammary metabolic response to altered nutritional state was also unlikely to be closely controlled by neural pathways. Severance of the cutaneous branch of the posterior division of the femoral nerve innervating the inguinal mammary glands did not reduce the high glucose uptake by mammary glands of either fed or refed rats, nor did denervation change the low glucose uptake by mammary glands of food-deprived rats. Denervation reduced blood flow in the associated mammary gland, however, indicating that neural pathways may play a role in supporting mammary blood flow when food is available. In in vitro experiments, the rate of glucose uptake was 35% lower in mammary acini from food-deprived rats than in fed rats 2.5 h after tissue removal, indicating some persistence of the food deprivation-induced suppression of mammary metabolism. Administration of insulin increased glucose uptake in acini from both fed and food-deprived rats, indicating that insulin may be involved in signaling the mammary gland of the restoration of nutrient supply when food-deprived rats are refed. The effects of administration of a gut extract in vivo and in vitro are discussed.     

Short communication: change in plasma ghrelin in dairy cows following an intravenous glucose challenge

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor and a potent orexigenic (appetite-stimulating) agent in humans and rodents, but little is known about its effect in dairy cows. Ten multiparous dairy cows 35 d in milk were subjected to an i.v. glucose challenge (300 mg of d-glucose/kg of body weight). Before infusion and at regular intervals after infusion, plasma glucose, insulin, nonesterified fatty acids (NEFA), growth hormone, epinephrine, and ghrelin concentrations were monitored. Plasma insulin rose (27.2 mU/L at 10 min) and NEFA, epinephrine, and ghrelin declined (nadir = 0.22 mmol/L, 22.2 μg/L, and 272 μg/L at 31, 13, and 22 min, respectively) after the glucose infusion. Ghrelin declined for 22 min before returning to suprabasal levels at approximately 75 min postinfusion. Sequential changes of the hormones and metabolites suggested a glucose transporter, type 2- and glucose transporter, type 4-mediated disposal of glucose, and an insulin-mediated reduction in NEFA. Ghrelin and epinephrine declined after glucose infusion and before the insulin peak, but the effect of insulin as a controlling factor in the hyperglycemic reduction in these hormones cannot be discounted. The post-nadir surge in ghrelin may be regulated by the decline in circulating concentrations of glucose and NEFA (an energy-deficit signal). The profile of change in plasma ghrelin in lactating dairy cows after a glucose challenge was similar to that in monogastric animals.     

Effects of intravenous glucose infusion and nutritional balance on serum concentrations of nonesterified fatty acids, glucose, insulin, and progesterone in nonlactating dairy cows

The objective of this study was to evaluate serum concentrations of nonesterified fatty acids, glucose, insulin, and progesterone in nonlactating dairy cows according to nutritional balance and glucose infusion. Ten nonlactating, ovariectomized Gir × Holstein cows were stratified by body weight (BW) and body condition score (BCS) on d −28 of the study, and randomly assigned to 1) negative nutrient balance (NB) or 2) positive nutrient balance (PB). From d −28 to d 0, cows were allocated according to nutritional treatment (5 cows/treatment) into 2 low-quality pastures with reduced forage availability. However, PB cows individually received, on average, 3 kg/cow per day (as-fed) of a concentrate during the study. All cows had an intravaginal progesterone releasing device inserted on d −14, which remained in cows until the end of the study. Cow BW and BCS were assessed again on d 0. On d 0, cows within nutritional treatment were randomly assigned to receive, in a crossover design containing 2 periods of 24 h each, 1) intravenous glucose infusion (GLU; 0.5 g of glucose/kg of BW, as a 5% glucose solution administered, on average, at 32 mL/min over a 3-h period), or 2) intravenous saline infusion (SAL; 0.9% solution infused on average at 32 mL/min over a 3-h period). Prior to the beginning of each period, all cows were fasted for 12 h. Blood samples were collected, relative to the beginning of the infusion, at −12 and −11.5 h (beginning of fasting), and at −0.5, 0, 0.5, 1, 2, 3, 4, 5, and 6 h. Following the last blood collection of period 1, cows received (PB) or not (NB) concentrate and were returned to their respective pastures. Changes in BCS and BW were greater in NB cows compared with PB cows (−0.60 and −0.25 ± 0.090 for BCS, respectively; −22.4 and 1.2 ± 6.58 kg for BW, respectively). Cows receiving GLUC had greater glucose concentrations from 0.5 to 3 h relative to infusion compared with SAL cows. Insulin concentrations were greater in PB cows assigned to GLUC compared with SAL cohorts at 0.5 and 3 h following infusion, whereas NB cows assigned to GLUC had greater insulin concentrations compared with SAL cohorts at 0.5, 1, 2, and 3 h. Progesterone concentrations were greater in PB cows assigned to GLUC at 2, 3, and 4 h following infusion compared with SAL cohorts. In conclusion, the effects of glucose infusion on serum concentrations of insulin and progesterone in nonlactating dairy cows were dependent on cow nutritional status.     

Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes

In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 μM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 μM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 cells.     

Acute experimental mastitis is not causal toward the development of energy-related metabolic disorders in early postpartum dairy cows

Twenty Holstein cows in early lactation (7 d in milk) were administered 100 μg of Escherichia coli lipopolysaccharide (LPS) dissolved in 10 mL of sterile 0.9% NaCl saline (treatment; TRT) or 10 mL of sterile saline (control) into both right mammary quarters to test the hypothesis that acute experimental mastitis would have negative impacts on aspects of energy metabolism that might lead to the development of metabolic disorders. A primed continuous intravenous infusion (14-μmol/kg of BW priming dose; 11.5-μmol/kg of BW per h continuous infusion) of 6,6-dideuterated glucose was used to determine pre- and posttreatment glucose kinetics using steady-state tracer methodologies. The LPS-treated cows displayed productive, clinical, and physiological signs of moderate to severe inflammation; control cows displayed no signs of immune activation. Pretreatment glucose rates of appearance (Ra) into plasma were similar (715 and 662 ± 33 mmol/h for TRT and control, respectively) between treatment groups. Intramammary LPS infusion into TRT cows resulted in increased glucose Ra relative to control cows (mean glucose Ra from 150 through 270 min after intramammary infusion were 815 and 674 ± 21 mmol/h for TRT and control cows, respectively). Furthermore, plasma concentrations of glucose increased, whereas plasma nonesterified fatty acids, glycerol, and β-hydroxybutyrate concentrations decreased, in TRT relative to control cows. Interestingly, plasma insulin concentration increased dramatically in TRT cows and occurred prior to the small increase in plasma glucose concentration. Although these results only represent the early stages of inflammation, they are not consistent with a causal relationship between mastitis and energy-related metabolic disorders and instead suggest a coordinated protective effect by the immune system on metabolism during the early stages of mammary insult.     

The effects of increasing doses of 2 preparations of long-acting insulin on short-term plasma profiles of glucose and insulin in lactating dairy cows

Two experiments were conducted to investigate effects of administering increasing doses of 2 different preparations of long-acting insulin on the 24-h profiles of plasma glucose and insulin concentrations in mid lactation dairy cows. The 2 separately analyzed experiments investigated the effects administering either Humulin N (H), a neutral protamine Hagedorn insulin, or insulin glargine (Lantus, L), an insulin analog, at doses of 0 (control), 0.1, 0.2, and 0.4 IU/kg of body weight in a randomized complete block design. Sixteen cows (237 ± 11 d in milk for H; 213 ± 10 d in milk for L; mean ± SD) were used for each insulin preparation, resulting in n = 4 for each dose within insulin preparation. Cows were fitted with a single jugular catheter on the day before the study. On the day of the study, cows were given treatments by subcutaneous injection of either sterile water or the designated insulin type and dose. Blood samples were taken hourly from the jugular catheter. Subcutaneous injection of both H and L resulted in linear decreases in plasma glucose concentrations, increased area under the curve, and decreased nadir for plasma glucose following administration of the insulin preparations. Plasma insulin concentration linearly increased with increasing dose of H. Though elevated concentrations of insulin were measurable in cows treated with H, they were not measurable in cows treated with L. Attempts to measure overall insulin concentrations and metabolites of L by a commercially available ELISA and a commercially available RIA kit were not successful and did not retrieve values that we felt truly represented the amount of insulin activity exhibited during this treatment. Both long-acting insulin preparations elicited insulin-like activity in lactating dairy cows, as evidenced by reduced plasma glucose concentrations. Given these results, the potential exists to use both H and L to study the effects of insulin in mid lactation dairy cows without the confounding effect of severe hypoglycemia (< 20 mg/dL) or concurrent provision of glucose during treatment.     

Effects of dietary energy and protein density on plasma concentrations of leptin and metabolic hormones in dairy heifers

The hormonal and metabolic signals that communicate the level of body energy reserves to the reproductive-mammary axis remain undefined in dairy cattle; consequently, our hypothesis was that leptin may fulfill this role. Our objectives were to determine the effects of diets differing in energy and protein density on dry matter intake (DMI), growth traits [body weight (BW), body condition score (BCS), back-fat (BF) thickness], and temporal changes in plasma concentrations of leptin, insulin, growth hormone (GH), insulin-like growth factor-1 (IGF-1), glucose, and nonesterified fatty acids (NEFA) in dairy heifers during the pre- and postpubertal periods. In period 1, heifers were randomly allotted (n = 10/diet) at 103 kg of BW to diets for a predicted average daily gain of 1.10 (high, H), 0.80 (medium, M), or 0.50 kg/d (low, L). Five heifers in each of the H and L groups were further studied during period 2, either at 12 mo of age (HA, LA) or at 330 kg of BW (HW, LW). The data provide evidence that 1) DMI (18%), BW (17%), and BF (5%) together explained 40% of the variation in plasma leptin concentrations (r² = 0.396); 2) unlike the acute postprandial increase in plasma insulin as a result of increased nutrient density (H 1.42 ± 0.09, M 1.02 ± 0.09, L 0.68 ± 0.11 ng/mL), plasma leptin concentrations did not respond acutely with a distinct postprandial profile; 3) although plasma leptin concentrations increased with age, leptin at puberty did not differ among treatment groups (H 5.63 ± 2.48, M 4.28 ± 0.55, L 4.12 ± 0.72 ng/mL) and there was no evidence of an abrupt transition in prepubertal plasma leptin concentrations; 4) plasma leptin concentrations may not be a critical trigger for puberty in rapidly growing heifers, but are apparently essential for puberty in heifers with normal or restricted growth rates; and 5) plasma concentrations of insulin (H 0.59 ± 0.07, M 0.43 ± 0.09, L 0.30 ± 0.09 ng/mL), IGF-1 (H 151.08 ± 16.47, L 82.51 ± 17.47 ng/mL), and glucose (H 81.35 ± 3.39, M 73.59 ± 2.34, L 68.25 ± 3.39 mg/dL) reflected nutrient density, whereas GH (H 1.82 ± 0.23, L 5.87 ± 0.45 ng/mL) and NEFA (H 209.54 ± 50.83, L 234.93 ± 48.97 μM) were inversely related to the plane of nutrition. Collectively, these data suggest that plasma concentrations of leptin may play a role in long-term regulation of energy reserves and puberty in growing Holstein heifers.     

Effects of chromium propionate on response to an intravenous glucose tolerance test in growing Holstein heifers

To test the effect of chromium propionate on glucose utilization in growing dairy heifers, 0, 5, 10, or 15 mg of chromium/d were fed to 20 Holstein heifers of 11 to 14 mo of age, in a replicated Latin square. A 2-wk adaptation period was followed by 4 periods of 2 wk each with a 2-wk flush out period between treatments. Treatments were allotted to periods in a design balanced for potential carryover effects. Chromium propionate was fed in 0.25 kg/d of ground corn individually. After 14 d on each treatment, animals were fitted with an indwelling jugular catheter, and an intravenous glucose tolerance test was conducted the following morning. Body weights increased throughout the experiment, but weights and condition scores were unaffected by treatment. Chromium supplementation increased basal glucose and decreased basal insulin and nonesterified fatty acids (NEFA) in serum in a dose-dependent, quadratic manner. Chromium increased glucose clearance rate as measured by half-life, time to nadir, and area under the curve. Over all periods, insulin concentrations tended to be lower in treated animals whereas clearance rates were unchanged. Serum NEFA levels were negatively correlated with glucose, such that treated animals with increased glucose had lower NEFA overall. There was an apparent long-term effect of chromium, because heifers in period 4 on the control diet had reduced insulin concentrations than those in the other control periods. Chromium propionate may increase glucose utilization in growing dairy heifers.     

Reduction of plasma NEFA concentration by nicotinic acid enhances the response to insulin in feed-restricted Holstein cows

The objective was to investigate the relationship between elevated plasma nonesterified fatty acid (NEFA) concentration and insulin resistance in Holstein cows. Six nonlactating, nongestating, ruminally cannulated Holstein cows were blocked by body condition score and randomly assigned to a sequence of 2 treatments in a crossover design. Cows were offered legume and grass hay ad libitum supplemented with minerals and vitamins and were allowed free access to water and a trace mineralized salt block. Mobilization of body reserves was stimulated by withdrawing forage for 48 h before initiation of treatments. Treatments consisted of 11 hourly abomasal infusions of water (control) or nicotinic acid (NA; 6 mg/h per kg of body weight) as an antilipolytic agent. Infusions of NA decreased plasma NEFA concentration from 545 μEq/L to approximately 100 μEq/L within 2 h after initiation of treatments, and differences were maintained throughout infusions. Intravenous glucose tolerance test was performed 8 h after initiation of treatments and was followed by 3 h of blood sampling. The reduction of plasma NEFA concentration led to significantly greater glucose clearance rate (1.9 vs. 1.2%/min) and to decreased glucose half-life (37 vs. 58 min), time to reach basal concentration (81 vs. 114 min) and glucose response area under the curve during 180 min of sampling [6,942 vs. 10,085 (μIU/mL) × 180 min]. Enhanced glucose clearance was achieved when plasma NEFA was reduced by NA, despite lower insulin concentration (70.0 vs. 97.9 ± 13.4 μIU/mL) and a tendency for smaller insulin response area under the curve during 180 min of sampling [7,646 vs. 12,104 ± 2,587 (μIU/mL) × 180 min], reflecting an increased response to endogenous insulin. Based on literature, we do not expect NA to have altered glucose metabolism directly; therefore, this experiment demonstrates a cause and effect relationship between elevated NEFA and insulin resistance in Holstein cows.     

Effects of abomasal infusion of linseed oil on responses to glucose and insulin in holstein cows

The objective was to study the effects of abomasal infusion of linseed oil, a source rich in n-3 C18:3, on whole-body response to insulin (experiment 1) and on insulin antilipolytic effects during feed restriction (experiment 2). In experiment 1, eight nonlactating, non-gestating cows were assigned to a crossover design, fed to meet maintenance requirements, and infused abomasally with either linseed oil (LIN) or tallow (TAL) at a rate of 0.54 g/kg of body weight per d for 5.5 d. Infusions were performed every 8 h during the first 3 d of each period and every 4 h thereafter. Intravenous glucose tolerance tests (IVGTT) were performed on d 5 of each period, followed by i.v. insulin challenges (IC) 12 h later. In experiment 2, six nonlactating, nongestating cows were assigned to a replicated 3 × 3 Latin square design. The experimental protocol included a water (WTR) treatment and feeding was suspended on d 3, leading to 50 and 62 h of feed restriction before IVGTT and IC, respectively. Clearance of glucose during IVGTT and IC was not affected by treatments in either experiment. However, LIN had an insulin sensitizing effect in experiment 1, because a lower insulin concentration led to the same clearance of glucose as TAL. In experiment 1, plasma nonesterified fatty acid (NEFA) concentration was low, reflecting a postprandial state, but NEFA was greater for LIN than TAL during IVGTT (108 vs. 88 ± 4 μEq/L) and IC (133 vs. 83 ± 9 μEq/L). In experiment 2, insulin concentrations during IVGTT did not differ across treatments. Basal plasma NEFA concentration before IVGTT tended to be greater for LIN than for TAL (612 vs. 508 μEq/L). Plasma NEFA clearance rate during IVGTT was greater for LIN than for TAL (2.8 vs. 2.5%/min), leading to a shorter time to reach half NEFA concentration (25 vs. 29 min) and greater absolute value of NEFA response area under the curve [AUC; −64,150 vs. −46,402 (μEq/L) × 180 min]. Data suggest that LIN enhanced the antilipolytic effects of insulin. Yet, other factors could have been involved because plasma NEFA concentration before IVGTT was 104 μEq/L greater for LIN than TAL for unknown reasons.     

Indian culinary plants enhance glucose-induced insulin secretion and glucose consumption in INS-1 β-cells and 3T3-L1 adipocytes

Six Indian plants, commonly used as culinary plants, herbs or spices (kikar; jamun; neem; harad; fenugreek; bitter gourd), were screened and compared for their antidiabetic potential in vitro. Aqueous plant extracts were prepared and assessed for their effect on the insulin secretion activity of rat pancreatic INS-1 β-cells and glucose consumption in mouse 3T3-L1 adipocytes in order to study their specific mechanisms of action. The effect of the plant extract concentration (25–1000 μg/ml) on insulin release and glucose consumption was also studied. All the extracts had a significant stimulatory effect on the insulin secretion of INS-1 cells. In the presence of kikar extract (100 μg/ml), an increase of 228% in insulin release was recorded compared to the control (5.6 mM glucose) whereas that was 270% and 367% in the presence of kikar and jamun extracts (500 μg/ml), respectively. 3T3-L1 cells treated with jamun extract (100 μg/ml) exhibited the highest increase in glucose consumption by the cells (94%, compared with the control) followed by harad (53%) and fenugreek (50%) extracts. A significant inhibitory effect of the fenugreek, kikar and jamun extracts on glucose diffusion across a dialysis membrane suggested that these extracts could partly act by decreasing glucose absorption in the small intestine. The results showed that a combination of these plants in diet could help in the management of both type 1 and type 2 diabetes.     

Effects of wortmannin,sodium nitroprusside,insulin, genistein,and guanosine triphosphate on chemotaxis and cell growth of Entodinium caudatum, Epidinium caudatum, and mixed ruminal protozoa

The mechanisms by which ruminal protozoa sense and migrate toward nutrients are not fully understood. Chemotaxis by many diverse eukaryotic cells is mediated by phosphatidylinositol-3-kinase, which is highly conserved in receptor tyrosine kinase (RTK) signaling pathways and consistently inhibited by wortmannin. In experiment 1a, increasing the concentration of wortmannin inhibited cell growth nonlinearly at 24 h of a culture of the rumen protozoan Entodinium caudatum, but high variability prevented growth inhibition of Epidinium caudatum from reaching significance. In experiment 1b, increasing the insulin concentration recovered 24-h cell counts for both cultures, depending on wortmannin concentration. In experiment 2, addition of sodium nitroprusside (Snp; activator of protein kinase G for cilial beat reversal in nonrumen ciliate models) at 500 µM or wortmannin at 200 µM in beakers containing rumen fluid decreased random swimming by mixed entodiniomorphids into capillary tubes (inserted into beakers) containing saline. Both Snp and wortmannin increased chemotaxis into tubes containing glucose compared with the beaker control. For isotrichids, beaker treatments had no response. Glucose increased chemotaxis, but peptides decreased chemotaxis even when combined with glucose. In experiment 3, we assessed preincubation of genistein (a purported RTK blocker in nonrumen ciliate models) at 40 or 400 µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100 µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100 µM reduced chemotaxis toward glucose for isotrichids. After the animal is fed, isotrichids that are depleted in glycogen migrate to the dorsal area of the rumen, and the rapid uptake of sugars is enhanced through strong chemotaxis but can be reversed by peptides or GTP. In contrast, entodiniomorphids are less intensely chemoattracted to glucose than isotrichids but are chemoattracted to peptides. Entodiniomorphids’ chemoattraction appears to be integrated with slower but prolonged availability of energy from digesting starch and fiber.     

miR-27a controls triacylglycerol synthesis in bovine mammary epithelial cells by targeting peroxisome proliferator-activated receptor gamma

Growing evidence has revealed that microRNA are central elements in milk fat synthesis in mammary epithelial cells. A negative regulator of adipocyte fat synthesis, miR-27a has been reported to be involved in the regulation of milk fat synthesis in goat mammary epithelial cells; however, the regulatory role of miR-27a in bovine milk fat synthesis remains unclear. In the present study, primary bovine mammary epithelial cells (BMEC) were harvested from mid-lactation cows and cultured in Dulbecco's modified Eagle's medium/F-12 medium with 10% fetal bovine serum, 5 μg/mL of insulin, 1 μg/mL of hydrocortisone, 2 μg/mL of prolactin, 1 μg/mL of progesterone, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. We found that the overexpression of miR-27a significantly suppressed lipid droplet formation and decreased the cellular triacylglycerol (TAG) levels, whereas inhibition of miR-27a resulted in a greater lipid droplet formation and TAG accumulation in BMEC. Meanwhile, overexpression of miR-27a inhibited mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein beta (C/EBPβ), perilipin 2 (PLIN2), and fatty acid binding protein 3 (FABP3), whereas miR-27a downregulation increased PPARG, C/EBPβ, FABP3, and CCAAT enhancer binding protein alpha (C/EBPα) mRNA expression. Furthermore, Western blot analysis revealed the protein level of PPARG in miR-27a mimic and inhibitor transfection groups to be consistent with the mRNA expression response. Moreover, luciferase reporter assays verified that PPARG was the direct target of miR-27a. In summary, these results indicate that miR-27a has the ability to control TAG synthesis in BMEC via targeting PPARG, suggesting that miR-27a could potentially be used to improve beneficial milk components in dairy cows.     

Insulin resistance in divergent strains of Holstein-Friesian dairy cows offered fresh pasture and increasing amounts of concentrate in early lactation

The objective of this study was to determine whether the physiological response to an intravenous glucose challenge would be affected by genetic strain or concentrate supplementation in grazing Holstein-Friesian cows in early lactation. North American (NA; n = 30) or New Zealand (NZ; n = 30) cows were randomly allocated to 1 of 3 feeding treatments. All cows were offered a generous pasture allowance, and 4 of the 6 groups received either 3 or 6 kg of dry matter (DM)/cow per day of concentrates. During wk 5 of lactation, all cows underwent an intravenous glucose challenge. Cows of NA origin produced more milk than NZ cows, but there was no significant strain effect on milk fat or protein yield. Milk yield and the yield of individual components increased with increasing level of concentrate eaten, but there were no significant strain × diet interactions. During wk 1 to 6, mean body weight and body condition score decreased in all treatments. Average body weight was greater in NA cows, but body condition score was greater for NZ cows. There was no strain or diet effect on the length of the postpartum anovulatory interval, with cows ovulating before 40 d postpartum on average. Glucose fractional turnover rate was greater in NZ cows compared with those of NA origin and in all cows receiving 6 kg of DM concentrates, indicating a less severe insulin resistance in those treatments. Consistent with this, the time taken to dispose of half the peak glucose concentration was less when 6 kg of DM concentrate was fed, and tended to be less in NZ than in NA cows. There was no effect of genetic strain on glucose area under the curve (AUC) at 60 or 120 min, but AUC at both time points was less in cows receiving 6 kg of DM concentrates per day. Neither genetic strain nor nutrition affected basal or peak insulin concentrations, insulin increment, or insulin AUC, and there were no strain × diet interactions for any of the glucose challenge response variables measured. In conclusion, differences in milk production between NA and NZ cows in early lactation can, at least in part, be explained by the greater degree of insulin resistance in the NA cows, and this insulin resistance can be overcome by supplementing grazing cows with 6 kg of DM concentrates.     

Effect of abomasal glucose infusion on splanchnic and whole-body glucose metabolism in periparturient dairy cows

Six periparturient Holstein cows fitted with ruminal cannulas and permanent indwelling catheters in the hepatic portal vein, hepatic vein, mesenteric vein, and an artery were used to study the effects of abomasal glucose infusion on splanchnic and whole-body glucose metabolism. The experimental design was a split plot, with cow as the whole plot, treatment as the whole-plot factor, and days in milk (DIM) as the subplot factor. Cows were assigned to 1 of 2 treatments: the control (no infusion) or infusion (1,500 g/d of glucose infused into the abomasum from the day of calving). Cows were sampled at 12 d prepartum and at 4, 15, and 29 DIM. To study portal-drained visceral uptake of arterial glucose, [U-¹³C]glucose was continuously infused into the jugular vein on sampling days. Postpartum, voluntary dry matter intake and milk yield increased at a lower rate with the infusion compared with the control. The net portal flux of glucose increased with the infusion compared with the control, and 67 ± 5% of the infused glucose was recovered as increased portal flux of glucose. The net hepatic flux of glucose was lower with the infusion compared with the control; however, the net hepatic flux of glucose per kilogram of dry matter intake was not affected by treatment. The arterial concentrations of glucose and insulin decreased and concentrations of nonesterified fatty acids increased from prepartum to 4 DIM with the control, but these effects were not observed with the infusion. The arterial concentration of β-hydroxybutyrate decreased more from prepartum to 4 DIM with the infusion, compared with the control. Uptake of arterial [U-¹³C]glucose in the portal-drained viscera was affected neither by the infusion nor by the DIM and averaged 2.5 ± 0.2%. The whole-body glucose supply changed to be less dependent on the recycling of lactate (Cori cycle) with the infusion. It was concluded that small intestinal glucose absorption is an efficient source of glucose to the peripheral tissues of dairy cows in very early lactation. At least 67% of the available glucose was recovered in the portal vein without affecting hepatic gluconeogenesis. Infused cows produced less milk and had a lower feed intake, indicating that an improved glucogenic status in very early lactation impaired metabolic adaptations to lactation.