Glucagon-like Peptide-2 Acutely Enhances Chylomicron Secretion in Humans Without Mobilizing Cytoplasmic Lipid Droplets.

CONTEXT: A portion of ingested fats are retained in the intestine for many hours before they are mobilized and secreted in chylomicron (CM) particles. Factors such as glucagon-like peptide-2 (GLP-2) and glucose can mobilize these stored intestinal lipids and enhance CM secretion. We have recently demonstrated in rodents that GLP-2 acutely enhances CM secretion by mechanisms that do not involve the canonical CM synthetic assembly and secretory pathways. OBJECTIVE: To further investigate the mechanism of GLP-2's potent intestinal lipid mobilizing effect, we examined intracellular cytoplasmic lipid droplets (CLDs) in intestinal biopsies of humans administered GLP-2 or placebo. DESIGN, SETTING, PATIENTS, AND INTERVENTIONS: A single dose of placebo or GLP-2 was administered subcutaneously 5 hours after ingesting a high-fat bolus. In 1 subset of participants, plasma samples were collected to quantify lipid and lipoprotein concentrations for 3 hours after placebo or GLP-2. In another subset, a duodenal biopsy was obtained 1-hour after placebo or GLP-2 administration for transmission electron microscopy and proteomic analysis. RESULTS: GLP-2 significantly increased plasma triglycerides by 46% (P = 0.009), mainly in CM-sized particles by 133% (P = 0.003), without reducing duodenal CLD size or number. Several proteins of interest were identified that require further investigation to elucidate their potential role in GLP-2-mediated CM secretion. CONCLUSIONS: Unlike glucose that mobilizes enterocyte CLDs and enhances CM secretion, GLP-2 acutely increased plasma CMs without significant mobilization of CLDs, supporting our previous findings that GLP-2 does not act directly on enterocytes to enhance CM secretion and most likely mobilizes secreted CMs in the lamina propria and lymphatics.

Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux.

Atherosclerosis is a chronic inflammatory condition in which macrophages play a major role. Janus kinase 2 (JAK2) is a pivotal molecule in inflammatory and metabolic signaling, and Jak2V617F activating mutation has recently been implicated with enhancing clonal hematopoiesis and atherosclerosis. To determine the essential in vivo role of macrophage (M)-Jak2 in atherosclerosis, we generate atherosclerosis-prone ApoE-null mice deficient in M-Jak2. Contrary to our expectation, these mice exhibit increased plaque burden with no differences in macrophage proliferation, recruitment or bone marrow clonal expansion. Notably, M-Jak2-deficient bone marrow derived macrophages show a significant defect in cholesterol efflux. Pharmacologic JAK2 inhibition with ruxolitinib also leads to defects in cholesterol efflux and accelerates atherosclerosis. Liver X receptor agonist abolishes the efflux defect and attenuates the accelerated atherosclerosis that occurs with M-Jak2 deficiency. Macrophages of individuals with the Jak2V617F mutation show increased efflux which is normalized when treated with a JAK2 inhibitor. Together, M-Jak2-deficiency leads to accelerated atherosclerosis primarily through defects in cholesterol efflux from macrophages.

Glucagon-like peptide-2 mobilizes lipids from the intestine by a systemic nitric oxide-independent mechanism.

AIM: To test the hypothesis that gut hormone glucagon-like peptide-2 (GLP-2) mobilizes intestinal triglyceride (TG) stores and stimulates chylomicron secretion by a nitric oxide (NO)-dependent mechanism in humans. METHODS: In a randomized, single-blind, cross-over study, 10 healthy male volunteers ingested a high-fat formula followed, 7 hours later, by one of three treatments: NO synthase inhibitor L-NG -monomethyl arginine acetate (L-NMMA) + GLP-2 analogue teduglutide, normal saline + teduglutide, or L-NMMA + placebo. TG in plasma and lipoprotein fractions were measured, along with measurement of blood flow in superior mesenteric and coeliac arteries using Doppler ultrasound in six participants. RESULTS: Teduglutide rapidly increased mesenteric blood flow and TG concentrations in plasma, in TG-rich lipoproteins, and most robustly in chylomicrons. L-NMMA significantly attenuated teduglutide-induced enhancement of mesenteric blood flow but not TG mobilization and chylomicron secretion. CONCLUSIONS: GLP-2 mobilization of TG stores and stimulation of chylomicron secretion from the small intestine appears to be independent of systemic NO in humans.

Oral Glucose Mobilizes Triglyceride Stores From the Human Intestine.

BACKGROUND & AIMS: The small intestine regulates plasma triglyceride (TG) concentration. Within enterocytes, dietary TGs are packaged into chylomicrons (CMs) for secretion or stored temporarily in cytoplasmic lipid droplets (CLDs) until further mobilization. We and others have shown that oral and intravenous glucose enhances CM particle secretion in human beings, however, the mechanisms through which this occurs are incompletely understood. METHODS: Two separate cohorts of participants ingested a high-fat liquid meal and, 5 hours later, were assigned randomly to ingest either a glucose solution or an equivalent volume of water. In 1 group (N = 6), plasma and lipoprotein TG responses were assessed in a randomized cross-over study. In a separate group (N = 24), duodenal biopsy specimens were obtained 1 hour after ingestion of glucose or water. Ultrastructural and proteomic analyses were performed on duodenal biopsy specimens. RESULTS: Compared with water, glucose ingestion increased circulating TGs within 30 minutes, mainly in the CM fraction. It decreased the total number of CLDs and the proportion of large-sized CLDs within enterocytes. We identified 2919 proteins in human duodenal tissue, 270 of which are related to lipid metabolism and 134 of which were differentially present in response to glucose compared with water ingestion. CONCLUSIONS: Oral glucose mobilizes TGs stored within enterocyte CLDs to provide substrate for CM synthesis and secretion. Future studies elucidating the underlying signaling pathways may provide mechanistic insights that lead to the development of novel therapeutics for the treatment of hypertriglyceridemia.

Impact of bariatric surgery on apolipoprotein C-III levels and lipoprotein distribution in obese human subjects.

BACKGROUND: Elevated apolipoprotein C-III (apoC-III) has been postulated to contribute to the atherogenic dyslipidemia seen in obesity and insulin-resistant states, mainly by impairing plasma triglyceride-rich lipoprotein (TRL) metabolism. Bariatric surgery is associated with improvements of several obesity-associated metabolic abnormalities, including a reduction in plasma triglycerides (TGs) and an increase in plasma high-density lipoprotein cholesterol (HDL-C). OBJECTIVES: We investigated the specific effect of bariatric surgery on apoC-III concentrations in plasma, non-HDL, and HDL fractions in relation to lipid profile parameters evolution. METHODS: A total of 132 obese subjects undergoing bariatric surgery, gastric bypass (n = 61) or sleeve gastrectomy (n = 71), were studied 1 month before surgery and 6 and 12 months after surgery. RESULTS: Plasma apoC-III, non-HDL-apoC-III, and HDL-apoC-III concentrations were markedly reduced after surgery and strongly associated with reduction in plasma TG. This decrease was accompanied by a redistribution of apoC-III from TRL to HDL fractions. In multivariate analysis, plasma apoC-III was the strongest predictor of TG reduction after surgery, and the increase of HDL-C was positively associated with plasma adiponectin and negatively with body mass index. CONCLUSION: Marked reduction of apoC-III and changes in its distribution between TRL and HDL consistent with a better lipid profile are achieved in obese patients after bariatric surgery. These apoC-III beneficial modifications may have implications in dyslipidemia improvement and contribute to cardiovascular risk reduction after surgery.

Supranutritional selenium intake from enriched milk casein impairs hepatic insulin sensitivity via attenuated IRS/PI3K/AKT signaling and decreased PGC-1α expression in male Sprague-Dawley rats.

Selenium (Se)-enriched milk provides antioxidant benefits and has therapeutic potential against cancer. However, both antidiabetic and prodiabetic effects have been attributed to Se. Our objective was to evaluate the effect of Se-enriched milk casein on insulin sensitivity in rats when given at the requirement of 0.25 ppm Se and supranutritionally on both low- and high-fat diets. Two hundred sixteen male Sprague-Dawley rats were fed low- or high-fat diets containing one, two or eight times the Se requirement in a randomized block design. After 7 weeks, 72 rats were subjected to the hyperinsulinemic-euglycemic clamp with [3-3H]glucose infusion to estimate glucose fluxes. Tissues were collected from the remaining 144 rats 8 min after ip saline or insulin injection. During hyperinsulinemic-euglycemic clamps, glucose infusion rate was 22% lower (P=.058), and endogenous glucose production was 76% higher (P=.054) when Se content increased from one to eight times the requirement on low-fat diets, indicating impaired hepatic insulin sensitivity. Se also decreased the ability for insulin to stimulate Akt phosphorylation at Thr308. Hepatic oxidation state and expression of selenoprotein P and glutathione peroxidase-1 were unaffected while expression of insulin receptor substrate (IRS)-1 and-2 and PPARγ coactivator-1α (PGC-1α) decreased with supranutritional Se and high-fat intake. In addition, hepatic expression of regulatory and catalytic subunits of phosphatidylinositol 3-kinase (PI3K) decreased with supranutritional intake of Se. Se intake from enriched casein up to eight times the requirement impairs hepatic insulin sensitivity in a mechanism similar to fat feeding, via attenuated IRS/PI3K/Akt signaling and decreased PGC-1α expression.

Nucleic Acid-Targeting Pathways Promote Inflammation in Obesity-Related Insulin Resistance.

Obesity-related inflammation of metabolic tissues, including visceral adipose tissue (VAT) and liver, are key factors in the development of insulin resistance (IR), though many of the contributing mechanisms remain unclear. We show that nucleic-acid-targeting pathways downstream of extracellular trap (ET) formation, unmethylated CpG DNA, or ribonucleic acids drive inflammation in IR. High-fat diet (HFD)-fed mice show increased release of ETs in VAT, decreased systemic clearance of ETs, and increased autoantibodies against conserved nuclear antigens. In HFD-fed mice, this excess of nucleic acids and related protein antigens worsens metabolic parameters through a number of mechanisms, including activation of VAT macrophages and expansion of plasmacytoid dendritic cells (pDCs) in the liver. Consistently, HFD-fed mice lacking critical responders of nucleic acid pathways, Toll-like receptors (TLR)7 and TLR9, show reduced metabolic inflammation and improved glucose homeostasis. Treatment of HFD-fed mice with inhibitors of ET formation or a TLR7/9 antagonist improves metabolic disease. These findings reveal a pathogenic role for nucleic acid targeting as a driver of metabolic inflammation in IR.

Effects of bariatric surgery on hepatic and intestinal lipoprotein particle metabolism.

PURPOSE OF REVIEW: Insulin resistance and type 2 diabetes, driven largely by obesity, are characterized by an increase in triglyceride-rich lipoproteins (TRLs) due to both reduced TRL clearance from the circulation and increased production by the liver (apoB-100 containing VLDLs) and intestine (apoB-48 containing chylomicrons). Bariatric surgery is the only treatment currently that leads to marked, sustained weight loss. Here, we will review the effects of bariatric surgery on circulating triglyceride/TRL and TRL production and clearance. RECENT FINDINGS: Bariatric surgery leads to a marked reduction in fasting and postprandial plasma triglyceride. Only one study to date has assessed TRL kinetics after bariatric surgery and has reported a reduction in TRL apoB-100 concentration (i.e. the number of VLDL particles) due to reduced production and increased clearance and reduced TRL apoB-48 concentration (the number of chylomicron particles) due to reduced production. Some bariatric surgery studies have reported no/weak correlation between weight loss and improvements in triglyceride/TRL, suggesting that as yet unidentified factors beyond weight loss may contribute to the marked changes in TRL that occur postbariatric surgery. SUMMARY: Available data suggest that bariatric surgery reduces triglyceride and intestinal and hepatic TRL production with increased clearance of hepatic TRL particles. These effects of bariatric surgery on TRL kinetics need to be confirmed with additional studies. Further studies are also needed to compare the effects of various bariatric surgery procedures on TRL kinetics and to elucidate underlying mechanisms.

Effects of bariatric surgery on hepatic and intestinal lipoprotein particle metabolism in obese, nondiabetic humans.

OBJECTIVE: The dyslipidemia of obesity and other insulin-resistant states is characterized by the elevation of plasma triglyceride-rich lipoproteins (TRL) of both hepatic (apoB-100-containing very low-density lipoprotein) and intestinal (apoB-48-containing chylomicrons) origin. Bariatric surgery is a well-established and effective modality for the treatment of obesity and is associated with improvements in several metabolic abnormalities associated with obesity, including a reduction in plasma triglycerides. Here, we have investigated the effect of bariatric surgery on TRL metabolism. APPROACH AND RESULTS: Twenty-two nondiabetic, obese subjects undergoing bariatric surgery: sleeve gastrectomy (n=12) or gastric bypass (n=10) were studied. Each subject underwent 1 lipoprotein turnover study 1 month before surgery followed by a second study, 6 months after surgery, using established stable isotope enrichment methodology, in constant fed state. TRL-apoB-100 concentration was significantly reduced after sleeve gastrectomy, explained by a decrease (P<0.05) in TRL-apoB-100 production rate and an increase (P<0.05) in TRL-apoB-100 fractional catabolic rate. TRL-apoB-48 concentration was also significantly reduced after sleeve gastrectomy, explained by reduction in TRL-apoB-48 production rate (P<0.05). For gastric bypass, although TRL-apoB-100 concentration declined after surgery (P<0.01), without a significant decline in TRL-apoB-48, there was no significant change in either TRL-apoB-100 or TRL-apoB-48 production rate or fractional catabolic rate. The reduction in TRL-apoB-100 concentration was significantly associated with a reduction in plasma apoC-III in the pooled group of patients undergoing bariatric surgery. CONCLUSIONS: This is the first human lipoprotein kinetic study to explore the mechanism of improvement of TRL metabolism after bariatric surgery. These effects may contribute to the decrease of cardiovascular mortality after surgery. CLINICAL TRIAL REGISTRATION URL: http://www.ClinicalTrials.gov. Unique identifier: NCT01277068.

New and emerging regulators of intestinal lipoprotein secretion.

Overproduction of hepatic apoB100-containing VLDL particles has been well documented in animal models and in humans with insulin resistance such as the metabolic syndrome and type 2 diabetes, and contributes to the typical dyslipidemia of these conditions. In addition, postprandial hyperlipidemia and elevated plasma concentrations of intestinal apoB48-containing chylomicron and chylomicron remnant particles have been demonstrated in insulin resistant states. Intestinal lipoprotein production is primarily determined by the amount of fat ingested and absorbed. Until approximately 10 years ago, however, relatively little attention was paid to the role of the intestine itself in regulating the production of triglyceride-rich lipoproteins (TRL) and its dysregulation in pathological states such as insulin resistance. We and others have shown that insulin resistant animal models and humans are characterized by overproduction of intestinal apoB48-containing lipoproteins. Whereas various factors are known to regulate hepatic lipoprotein particle production, less is known about factors that regulate the production of intestinal lipoprotein particles. Monosacharides, plasma free fatty acids (FFA), resveratrol, intestinal peptides (e.g. GLP-1 and GLP-2), and pancreatic hormones (e.g. insulin) have recently been shown to be important regulators of intestinal lipoprotein secretion. Available evidence in humans and animal models strongly supports the concept that the small intestine is not merely an absorptive organ but rather plays an active role in regulating the rate of production of chylomicrons in fed and fasting states. Metabolic signals in insulin resistance and type 2 diabetes and in some cases an aberrant intestinal response to these factors contribute to the enhanced formation and secretion of TRL. Understanding the regulation of intestinal lipoprotein production is imperative for the development of new therapeutic strategies for the prevention and treatment of dyslipidemia. Here we review recent developments in this field and present evidence that intestinal lipoprotein production is a process with metabolic plasticity and that modulation of intestinal lipoprotein secretion may be a feasible therapeutic strategy in the treatment of dyslipidemia and possibly prevention of atherosclerosis.

High-dose resveratrol treatment for 2 weeks inhibits intestinal and hepatic lipoprotein production in overweight/obese men.

OBJECTIVE: Overproduction of hepatic apolipoprotein B (apoB)-100 containing very low-density lipoprotein particles and intestinal apoB-48 containing chylomicrons contributes to hypertriglyceridemia seen in conditions such as obesity and insulin resistance. Some, but not all, preclinical and clinical studies have demonstrated that the polyphenol resveratrol ameliorates insulin resistance and hypertriglyceridemia. Here, we assessed intestinal and hepatic lipoprotein turnover, in humans, after 2 weeks of treatment with resveratrol (1000 mg daily for week 1 followed by 2000 mg daily for week 2) or placebo. APPROACH AND RESULTS: Eight overweight or obese individuals with mild hypertriglyceridemia were studied on 2 occasions, 4 to 6 weeks apart, after treatment with resveratrol or placebo in a randomized, double-blinded, crossover study. Steady-state lipoprotein kinetics was assessed in a constant fed state with a primed, constant infusion of deuterated leucine. Resveratrol treatment did not significantly affect insulin sensitivity (homeostasis model of assessment of insulin resistance), fasting or fed plasma triglyceride concentration. Resveratrol reduced apoB-48 production rate by 22% (P=0.007) with no significant effect on fractional catabolic rate. Resveratrol reduced apoB-100 production rate by 27% (P=0.02) and fractional catabolic rate by 26% (P=0.04). CONCLUSIONS: These results indicate that 2 weeks of high-dose resveratrol reduces intestinal and hepatic lipoprotein particle production. Long-term studies are needed to evaluate the potential clinical benefits of resveratrol in patients with hypertriglyceridemia, who have increased concentrations of triglyceride-rich lipoprotein apoB-100 and apoB-48. CLINICAL TRIAL REGISTRATION URL: www.clinicaltrials.gov. Unique identifier: NCT01451918.

Exenatide, a glucagon-like peptide-1 receptor agonist, acutely inhibits intestinal lipoprotein production in healthy humans.

OBJECTIVE: Incretin-based therapies for the treatment of type 2 diabetes mellitus improve plasma lipid profiles and postprandial lipemia, but their exact mechanism of action remains unclear. Here, we examined the acute effect of the glucagon-like peptide-1 receptor agonist, exenatide, on intestinal and hepatic triglyceride-rich lipoprotein production and clearance in healthy humans. METHODS AND RESULTS: Fifteen normolipidemic, normoglycemic men underwent 2 studies each (SC 10 µg exenatide versus placebo), 4 to 6 weeks apart, in random order, in which triglyceride-rich lipoprotein particle kinetics were examined with a primed, constant infusion of deuterated leucine and analyzed by multicompartmental modeling under pancreatic clamp conditions. A fed state was maintained during each study by infusing a high-fat, mixed macronutrient, liquid formula at a constant rate directly into the duodenum via a nasoduodenal tube. Exenatide significantly suppressed the plasma concentration and production rate of triglyceride-rich lipoprotein-apolipoprotein B-48, but not of triglyceride-rich lipoprotein-apolipoprotein B-100. CONCLUSIONS: These results suggest a possible direct effect of exenatide on intestinal lipoprotein particle production, independent of changes in weight gain and satiety as seen in long-term studies and independent of changes in gastric emptying. This finding expands our understanding of the effects of exenatide in metabolic regulation beyond its primary therapeutic role in regulation of glucose homeostasis. Clinical Trial Registration- URL: http://www.clinicaltrials.gov, NCT01056549.

C-reactive protein impairs hepatic insulin sensitivity and insulin signaling in rats: role of mitogen-activated protein kinases.

UNLABELLED: Plasma C-reactive protein (CRP) concentration is increased in the metabolic syndrome, which consists of a cluster of cardiovascular disease risk factors, including insulin resistance. It is not known, however, whether CRP is merely a marker of accompanying inflammation or whether it contributes causally to insulin resistance. The objective of this study is to investigate the role that CRP may play in the development of insulin resistance. We examined the effect of single-dose intravenous administration of purified human (h)CRP on insulin sensitivity in Sprague-Dawley rats using the euglycemic, hyperinsulinemic clamp technique. hCRP was associated with impaired insulin suppression of endogenous glucose production with no reduction in peripheral tissue glucose uptake, suggesting that hCRP mediated insulin resistance in the liver but not extrahepatic tissues. We further assessed components of the insulin signaling pathway and mitogen-activated protein kinases (MAPKs) in the liver. Liver tissues derived from hCRP-treated rats showed reduced insulin-stimulated insulin receptor substrate (IRS) tyrosine phosphorylation, IRS/phosphatidylinositol 3-kinase (PI3K) association, and Akt phosphorylation, consistent with hCRP-induced impairment of hepatic insulin signaling. Furthermore, hCRP enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK as well as IRS-1 Ser(612) . Finally, we observed in primary cultured rat hepatocytes that U0126 (a selective inhibitor of MAPK/ERK kinase1/2) corrected hCRP-induced impairment of insulin signaling. CONCLUSIONS: hCRP plays an active role in inducing hepatic insulin resistance in the rat, at least in part by activating ERK1/2, with downstream impairment in the insulin signaling pathway.

Enhanced cellular uptake of remnant high-density lipoprotein particles: a mechanism for high-density lipoprotein lowering in insulin resistance and hypertriglyceridemia.

A low level of high-density lipoprotein (HDL) cholesterol is characteristic of insulin resistance and hypertriglyceridemia and likely contributes to the increased risk of cardiovascular disease associated with these conditions. One pathway involves enhanced clearance of lipolytically modified HDL particles, but the underlying mechanisms remain poorly understood. Here, we examine the effect of triglyceride enrichment and hepatic lipase hydrolysis on HDL binding, internalization, and degradation in cultured liver and kidney cells. Maximal binding of remnant HDL (HDL enriched with triglycerides followed by hepatic lipase hydrolysis), but not binding affinity, was markedly higher than native and triglyceride-rich HDL in both HepG2 cells and HEK293 cells. Compared with native and triglyceride-rich HDL, remnant HDL was internalized to a greater extent in both cell types and was more readily degraded in HEK293 cells. The increased binding of remnant HDL was not mediated by the low-density lipoprotein receptor or scavenger receptor class B type I (SR-BI), because enhanced remnant HDL binding was observed in low-density lipoprotein receptor-deficient cells with or without SR-BI overexpression. Disruption of cell surface heparan sulfate proteoglycans or blockage of apolipoprotein E-mediated lipoprotein binding also did not abolish the enhanced remnant HDL binding. Our observations indicate that remodeling of triglyceride-enriched HDL by hepatic lipase may result in enhanced binding, internalization, and degradation in tissues involved in HDL catabolism, contributing to rapid clearance and overall lowering of plasma HDL cholesterol in insulin resistance and hypertriglyceridemia.

Description of glucose transport in isolated bovine mammary epithelial cells by a three-compartment model.

Initial rates of glucose entry into isolated bovine mammary epithelial cells display moderate degrees of asymmetry and cooperative interactions between export and import sites. The present study examined the hypothesis that these kinetic features are due to compartmentalization of intracellular glucose. Net uptake of 3-O-methyl-d-[1-(3)H]glucose (3-OMG) by isolated bovine mammary epithelial cells was measured at 37 degrees C. The time course of 3-OMG net uptake was better fitted by a double-exponential equation than by a single- or triple-exponential equation. Compartmental analysis of the time course curve suggested that translocated 3-OMG is distributed into two compartments with fractional volumes of 32.6 +/- 5.7% and 67.4 +/- 5.7%, respectively. The results support the view that glucose transport in bovine mammary epithelial cells is a multistep process consisting of two serial steps: fast, carrier-mediated, symmetric translocation of sugar across the cell plasma membrane into a small compartment and subsequent slow exchange of posttranslocated sugar between two intracellular compartments. A three-compartment model of this system successfully simulated the observed time course of 3-OMG net uptake and the observed dependence of unidirectional entry rates on intra- and extracellular 3-OMG concentrations. Simulations indicated that backflux of radiolabeled sugar from the small compartment to extracellular space during 15 s of incubation gives rise to the apparent asymmetry, trans-stimulation, and cooperativity of mammary glucose transport kinetics. The fixed-site carrier model overestimated the rate of glucose accumulation in cells, and its features can be accounted for by the compartmentalization of intracellular sugar.

Glucose transporter in bovine mammary epithelial cells is an asymmetric carrier that exhibits cooperativity and trans-stimulation.

Glucose transport kinetics were quantified in isolated bovine mammary epithelial cells using 3-O-methyl-D-glucose. Isolated cells retained satisfactory viability and glucose uptake activity, which was inhibited by cytochalasin B, phloretin, HgCl2, and low temperature. Initial rates of entry were measured over a 15-s interval at 37 degrees C under zero-trans, equilibrium-exchange, high-cis, and high-trans concentrations of 3-O-methyl-D-glucose between 0 and 20 mM. The combined set of rate measurements from all experimental conditions was fit to the fixed-site carrier model by nonlinear regression to estimate parameters of transport. For the regression between predicted and observed initial rates, r2 was 0.97. Forward Vmax was estimated at 18.2 nmol.min-1.mg protein-1, and the Michaelis constant was 8.29 mM. The cooperativity parameter was 1.63, trans-stimulation was 2.13-fold, and asymmetry was 2.06-fold. On the basis of the kinetic parameters, variations in intracellular glucose concentrations are not responsible for the range of glucose uptakes by bovine mammary glands observed in vivo.