Glucose metabolism and the somatotropic axis in dairy cows after abomasal infusion of essential fatty acids together with conjugated linoleic acid during late gestation and early lactation.

Sufficient glucose availability is crucial for exploiting the genetic potential of milk production during early lactation, and endocrine changes are mainly related to repartitioning of nutrient supplies toward the mammary gland. Long-chain fatty acids, such as essential fatty acids (EFA) and conjugated linoleic acid (CLA), have the potential to improve negative energy balance and modify endocrine changes. In the present study, the hypothesis that combined CLA and EFA treatment supports glucose metabolism around the time of calving and stimulates insulin action and the somatotropic axis in cows in an additive manner was tested. Rumen-cannulated German Holstein cows (n = 40) were investigated from wk 9 antepartum (AP) until wk 9 postpartum (PP). The cows were abomasally supplemented with coconut oil (CTRL, 76 g/d); 78 g/d of linseed and 4 g/d of safflower oil (EFA); Lutalin (CLA, isomers cis-9,trans-11 and trans-10,cis-12 CLA, each 10 g/d); or the combination of EFA+CLA. Blood samples were collected several times AP and PP to determine the concentrations of plasma metabolites and hormones related to glucose metabolism and the somatotropic axis. Liver tissue samples were collected several days AP and PP to measure glycogen concentration and the mRNA abundance of genes related to gluconeogenesis and the somatotropic axis. On d 28 AP and 21 PP, endogenous glucose production (eGP) and glucose oxidation (GOx) were measured via tracer technique. The concentration of plasma glucose was higher in CLA than in non-CLA-treated cows, and the plasma ß-hydroxybutyrate concentration was higher in EFA than in non-EFA cows on d 21 PP. The eGP increased from AP to PP with elevated eGP in EFA and decreased eGP in CLA-treated cows; GOx was lower in CLA than in CTRL on d 21 PP. The plasma insulin concentration decreased after calving in all groups and was higher in CLA than in non-CLA cows at several time points. Plasma glucagon and cortisol concentrations on d 21 PP were lower in CLA than non-CLA groups. The glucagon/insulin and glucose/insulin ratios were higher in CTRL than in CLA group during the transition period. Plasma IGF-I concentration was lower in EFA than non-EFA cows on d 42 AP and was higher during the dry period and early lactation in CLA than in non-CLA cows. The IGF binding protein (IGFBP)-3/-2 ratio in blood plasma was higher in CLA than in non-CLA cows. Hepatic glycogen concentration on d 28 PP was higher, but the mRNA abundance of PC and IGFBP2 was lower in CLA than non-CLA cows on d 1 PP. The EFA treatment decreased the mRNA abundance of IGFBP3 AP and PCK1, PCK2, G6PC, PCCA, HMGCS2, IGFBP2, and INSR at several time points PP. Results indicated elevated concentrations of plasma glucose and insulin along with the stimulation of the somatotropic axis in cows treated with CLA, whereas EFA treatment stimulated eGP but not mRNA abundance related to eGP PP. The systemic effects of the combined EFA+CLA treatment were very similar to those of CLA treatment, but the effects on hepatic gene expression partially corresponded to those of EFA treatment.

Effect of maternal supplementation with essential fatty acids and conjugated linoleic acid on metabolic and endocrine development in neonatal calves.

We tested the hypothesis that the maternal supply of essential fatty acids (EFA), especially α-linolenic acid, and conjugated linoleic acid (CLA), affects glucose metabolism, the endocrine regulation of energy metabolism and growth, and the intestinal development of neonatal calves. We studied calves from dams that received an abomasal infusion of 76 g/d coconut oil (CTRL; n = 9), 78 g/d linseed oil and 4 g/d safflower oil (EFA; n = 9), 38 g/d Lutalin (BASF SE) containing 27% cis-9,trans-11 and trans-10,cis-12 CLA (CLA; n = 9), or a combination of EFA and CLA (EFA+CLA; n = 11) during the last 63 d of gestation and early lactation. Calves received colostrum and transition milk from their own dam for the first 5 d of life. Insulin-like growth factor (IGF)-I, leptin, and adiponectin concentrations were measured in milk. Blood samples were taken before first colostrum intake, 24 h after birth, and from d 3 to 5 of life before morning feeding to measure metabolic and endocrine traits in plasma. On d 3 of life, energy expenditure was evaluated by a bolus injection of NaH13CO3 and determination of CO2 appearance rate. On d 4, additional blood samples were taken to evaluate glucose first-pass uptake and 13CO2 enrichment after [13C6]-glucose feeding and intravenous [6,6-2H2]-glucose bolus injection, as well as postprandial changes in glucose, nonesterified fatty acids (NEFA), insulin, and glucagon. On d 5, calves were killed 2 h after feeding and samples of small intestinal mucosa were taken for histomorphometric measurements. The concentrations of IGF-I, adiponectin, and leptin in milk decreased during early lactation in all groups, and the concentrations of leptin in first colostrum was higher in EFA than in CTRL cows. Plasma glucose concentration before first colostrum intake was higher in EFA calves than in non-EFA calves and was lower in CLA calves than in non-CLA calves. Plasma IGF-I concentration was higher on d 1 before colostrum intake in EFA calves than in EFA+CLA calves and indicated an overall CLA effect, with lower plasma IGF-I in CLA than in non-CLA calves. Postprandial NEFA concentration was lowest in EFA and CLA calves. The postprandial rise in plasma insulin was higher in EFA than in non-EFA calves. Plasma adiponectin concentration increased from d 1 to d 2 in all groups and was higher on d 3 in CLA than in non-CLA calves. Plasma leptin concentration was higher on d 4 and 5 in EFA than in non-EFA calves. Maternal fatty acid treatment did not affect energy expenditure and first-pass glucose uptake, but glucose uptake on d 4 was faster in EFA than in non-EFA calves. Crypt depth was lower, and the ratio of villus height to crypt depth was higher in the ilea of CLA than non-CLA calves. Elevated plasma glucose and IGF-I in EFA calves immediately after birth may indicate an improved energetic status in calves when dams are supplemented with EFA. Maternal EFA and CLA supplementation influenced postprandial metabolic changes and affected factors related to the neonatal insulin response.

Effects of a combined essential fatty acid and conjugated linoleic acid abomasal infusion on metabolic and endocrine traits, including the somatotropic axis, in dairy cows.

The objective of this study was to test the effects of essential fatty acids (EFA), particularly α-linolenic acid (ALA), and conjugated linoleic acid (CLA) supplementation on metabolic and endocrine traits related to energy metabolism, including the somatotropic axis, in mid-lactation dairy cows. Four cows (126 ± 4 d in milk) were used in a dose-escalation study design and were abomasally infused with coconut oil (CTRL; 38.3 g/d; providing saturated fatty acids), linseed and safflower oils (EFA; 39.1 and 1.6 g/d; n-6:n-3 FA ratio = 1:3), Lutalin (CLA; cis-9,trans-11 and trans-10,cis-12 CLA, 4.6 g/d of each), or EFA and CLA (EFA+CLA) for 6 wk. The initial dosage was doubled twice after 2 wk, resulting in 3 dosages (dosages 1, 2, and 3). Each cow received each fat treatment at different times. Cows were fed with a corn silage-based total mixed ration providing a low-fat content and a high n-6:n-3 fatty acid ratio. Plasma concentrations of metabolites and hormones (insulin-like growth factor-binding proteins only on wk 0 and 6) were analyzed at wk 0, 2, 4, and 6 of each treatment period. Liver biopsies were taken before starting the trial and at wk 6 of each treatment period to measure hepatic mRNA abundance of genes linked to glucose, cholesterol and lipid metabolism, and the somatotropic axis. The changes in the milk and blood fatty acid patterns and lactation performance of these cows have already been published in a companion paper. The plasma concentration of total cholesterol increased with dosage in all groups, except CLA, reaching the highest levels in EFA+CLA and CTRL compared with CLA. The high-density lipoprotein cholesterol plasma concentration increased in CTRL and was higher than that in EFA and CLA, whereas the concentration of low-density lipoprotein cholesterol increased in a dose-dependent manner in EFA and EFA+CLA, and was higher than that in CLA. Hepatic mRNA expression of 3-hydroxy-3-methyl-glutaryl-CoA synthase 1 was upregulated in all groups but was highest in EFA+CLA. Expression of sterol regulatory element-binding factor 1 tended to be lowest due to EFA treatment, whereas expression of long chain acyl-CoA-synthetase was lower in EFA than in CTRL. Hepatic mRNA expression of GHR1A tended to be higher in EFA+CLA than in CTRL. The plasma concentration of insulin-like growth factor I increased in CLA, and the plasma IGFBP-2 concentration was lower in EFA+CLA than in CTRL at wk 6. The plasma concentration of adiponectin decreased in EFA+CLA up to dosage 2. Plasma concentrations of albumin and urea were lower in CLA than in CTRL throughout the experimental period. Supplementation with EFA and CLA affected cholesterol and lipid metabolism and their regulation differently, indicating distinct stimulation after the combined EFA and CLA treatment. The decreased IGFBP-2 plasma concentration and upregulated hepatic mRNA abundance of GHR1A in EFA+CLA-supplemented cows indicated the beneficial effect of the combined EFA and CLA treatment on the somatotropic axis in mid-lactation dairy cows. Moreover, supplementation with CLA might affect protein metabolism in dairy cows.

Effects of colostrum instead of formula feeding for the first 2 days postnatum on whole-body energy metabolism and its endocrine control in neonatal calves.

Colostrum provides high amounts of nutritive and non-nutritive substrates, which are essential for calf nutrition and passive immunization. Colostral growth factors and hormones have beneficial effects on postnatal maturation and may affect substrate utilization and energy expenditure in neonatal calves. We tested the hypothesis that energy metabolism and its endocrine regulation differ during the first 10 d of life in calves fed either colostrum or a milk-based formula with a similar nutrient composition to colostrum, but largely depleted of bioactive substances, for the first 2 d postnatum. Male Holstein calves (n = 18) were fed either pooled colostrum (COL; n = 9) or a milk-based formula (FOR; n = 9) for the first 2 d of life. From d 3 on, all calves received same milk replacer. On d 2 and 7 of life, calves were placed in a respiration chamber for indirect calorimetric measurements to calculate heat production, fat (FOX) and carbohydrate oxidation (COX), as well as respiratory quotient. Blood was sampled on d 1 before first colostrum intake and on d 2, 3, 7, 8, 9, and 10 before morning feeding, to measure plasma concentrations of immunoglobulins, metabolites, and hormones. Additional postprandial blood samples were taken on d 1 and 9 at 30, 60, 120, 240, and 420 min after milk feeding. Liver samples were collected on d 10 of life to determine gene expression related to energy metabolism. Formula-fed calves showed lower plasma concentrations of total protein, immunoglobulins, haptoglobin, leptin, adiponectin, and insulin-like growth factor (IGF) binding protein (IGFBP)-4 during the whole study but temporarily higher plasma concentrations of urea, insulin, glucagon, triglyceride, and cholesterol on the first day after feeding, compared with concentrations in COL. The temporary increase in glucagon, triglyceride, and cholesterol on d 1 reversed on d 2 or 3, showing higher concentrations in COL than in FOR calves. In FOR, IGF-I, IGFBP-2, and IGFBP-3 were lower on d 3 than in COL. Interestingly, FOR calves had higher heat production during respiratory measurements on d 2 and higher body temperature on d 2, 3, and 5 than those of COL. The hepatic mRNA abundance of cytosolic phosphoenolpyruvate carboxykinase was higher in FOR than in COL. Our results indicate that first milk feeding after birth influenced whole-body energy expenditure but not FOX and COX in neonatal calves, and the absorption of colostral leptin and adiponectin might affect insulin sensitivity on d 1 of life.

Influence of ad libitum milk replacer feeding and butyrate supplementation on the systemic and hepatic insulin-like growth factor I and its binding proteins in Holstein calves.

Ad libitum milk feeding and butyrate (B) supplementation have the potential to stimulate postnatal growth and development in calves. The somatotropic axis is the main endocrine regulator of postnatal growth and may be affected by both ad libitum milk replacer (MR) feeding and B supplementation in calves. We hypothesized that ad libitum MR feeding and B supplementation stimulate systemic and hepatic insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP) in preweaning calves. Sixty-four (32 male, 32 female) Holstein calves were examined from birth until wk 11 of life. Calves received MR either ad libitum (Adl) or restrictively (6 L/d; Res). In each feeding group half of the calves received a MR with 0.24% butyrate and the other half received same MR without butyrate. Ad libitum MR feeding was performed from d 4 until wk 8 of age. From wk 9 to 10, Adl and Res calves were gradually weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. Feed intake was measured daily and body weight weekly. Blood samples for analyzing plasma concentrations of glucose, insulin, IGF-I, and IGFBP-2, -3, and -4 were taken on d 1, 2, 4, and 7, then weekly or every other week (IGFBP) until wk 11 of life. Liver samples were taken on d 50 and at the end of the study (d 80) to measure gene expression of the growth hormone receptor 1A (GHR1A), IGF1, IGFBP1 to 4, and of the IGF Type 1 and insulin receptor in the liver. Intake of MR and body weight were greater, but concentrate intake was lower in Adl than in Res. Plasma concentrations of IGF-I and IGFBP-3 were greater and plasma concentration of IGFBP-2 was lower in Adl than in Res during the ad libitum milk feeding period. After reduction of MR in both groups to 2 L/d plasma concentrations of IGF-I and IGFBP-4 were lower and plasma concentration of IGFBP-2 was higher in Adl than in Res. Supplementation of B depressed plasma IGF-I from wk 1 to 4 and in wk 9. On d 50, mRNA abundance of the GHR1A and IGF1 was greater and of IGFBP2 mRNA was lower in Adl than in Res. At d 80, IGFBP2 mRNA was greater in Adl than in Res, and IGFBP2 mRNA increased with B supplementation. Ad libitum MR feeding stimulated the systemic and hepatic IGF system and mirrored the greater growth rate during the ad libitum MR feeding, whereas butyrate supplementation partly reduced the systemic and hepatic IGF system.

Methylating micronutrient supplementation during pregnancy influences foetal hepatic gene expression and IGF signalling and increases foetal weight.

PURPOSE: Maternal diet during pregnancy impacts foetal growth and development. In particular, dietary levels of methylating micronutrients (methionine, folate, choline, vitamins B6, and B12) interfere with the availability and allocation of methyl groups for methylation reactions, thereby influencing normal transcription. However, the currently recommended methylating micronutrient supplementation regimen is haphazard and arbitrary at best. METHODS: To investigate the effects of a methylating micronutrient-rich maternal diet, pregnant Pietrain sows were fed either a standard diet (CON) or a diet supplemented with methionine, folate, choline, B6, B12, and zinc (MET). Foetal liver and muscle (M. longissimus dorsi) tissues were collected at 35, 63, and 91 days post-conception. Transcriptional responses to diet were assessed in foetal liver. Altered insulin-like growth factor (IGF) signalling in transcriptome analyses prompted investigation of IGF-2 and insulin-like growth factor binding proteins (IGFBPs) levels in muscle and liver. RESULTS: Maternal diet enriched with methylating micronutrients was associated with increased foetal weight in late gestation. Hepatic transcriptional patterns also revealed differences in vitamin B6 and folate metabolism between the two diets, suggesting that supplementation was effective. Additionally, shifts in growth-supporting metabolic routes of the lipid and energy metabolism, including IGF signalling, and of cell cycle-related pathways were found to occur in liver tissue in supplemented individuals. Weight differences and modulated IGF pathways were also reflected in the muscle content of IGF-2 (increased in MET) and IGFBP-2 (decreased in MET). CONCLUSIONS: Maternal dietary challenges provoke stage-dependent and tissue-specific transcriptomic modulations in the liver pointing to molecular routes contributing to the organismal adaptation. Subtle effects on late foetal growth are associated with changes in the IGF signalling mainly in skeletal muscle tissue that is less resilient to dietary stimuli than liver.

Antepartal insulin-like growth factor 1 and insulin-like growth factor binding protein 2 concentrations are indicative of ketosis in dairy cows.

A study involving a small number of cows found that the concentrations of insulin-like growth hormone 1 (IGF1) may be a useful predictor of metabolic disease. Further, IGF1 may provide also a pathophysiological link to metabolic diseases such as ketosis. The objective of the current study was to test whether the low antepartal total IGF1 or IGF1 binding protein (IGFBP) concentrations might predict ketosis under field conditions. Clinical examinations and blood sampling were performed antepartum (262-270 d after artificial insemination) on 377 pluriparous pregnant Holstein Friesian cows. The presence of postpartum diseases were recorded (ketosis, fatty liver, displacement of the abomasum, hypocalcemia, mastitis, retention of fetal membranes, and clinical metritis or endometritis), and the concentrations of IGF1, IGFBP2, IGFBP3, and nonesterified fatty acids were measured. Cows with postpartum clinical ketosis had lower IGF1 concentrations antepartum than healthy cows. The sensitivity of antepartal IGF1 as a marker for postpartum ketosis was 0.87, and the specificity was 0.43; a positive predictive value of 0.91 and a negative predictive value of 0.35 were calculated. The cows with ketosis and retained fetal membranes had lower IGFBP2 concentrations compared with the healthy cows. It can be speculated that lower IGF1 production in the liver during late pregnancy may increase growth hormone secretions and lipolysis, thereby increasing the risk of ketosis. Lower IGFBP2 concentrations may reflect the suppression of IGFBP2 levels through higher growth hormone secretion. In conclusion, compared with nonesterified fatty acids as a predictive parameter, IGF1 and IGFBP2 may represent earlier biomarkers of inadequate metabolic adaptation to the high energy demand required postpartum.

Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows.

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d -20, -10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply correspond in the brain. We thus assume independent control of IGFBP distribution between plasma and CSF. Due to the known anorexic effect of IGF-I, elevated plasma concentrations of IGFBP-2 and IGFBP-4 during the postpartum period in conjunction with reduced plasma IGF-I concentrations may be interpreted as an endocrine response against negative energy balance in early lactation in dairy cows.

Hepatic mRNA expression of acid labile subunit and deiodinase 1 differs between cows selected for high versus low concentrations of insulin-like growth factor 1 in late pregnancy.

The somatotropic axis is a key metabolic pathway during transition from late pregnancy to early lactation in dairy cows. The first objective of this study was to determine the feasibility of selecting cows with persistent differences in total insulin-like growth factor 1 (IGF-1) concentration by taking only a single antepartum blood sample. The second objective was to elucidate the underlying causes of differences in peripheral IGF-1 concentrations throughout late pregnancy and whether hormonal axes also differed in dairy cows with low versus high IGF-1. Twenty clinically healthy Holstein Friesian cows were chosen based on their plasma IGF-1 concentration at 244 to 254 d after artificial insemination (AI) and other selection criteria (health status, body condition score, number of lactations). These cows were selected from a large-scale farm, transported to the clinic, and monitored daily from 261 to 275 d after AI. The concentrations of IGF-1, growth hormone, IGF binding proteins 2, 3, and 4, insulin, cortisol, thyroid hormones, progesterone, and estradiol were measured. Ultimately, 7 IGF-1-low and 7 IGF-1-high cows were statistically analyzed. Additionally, a liver biopsy was taken on d 270 ± 1 after AI for analysis of gene expression of somatotropic family members, liver deiodinase 1, and suppressor of cytokine signaling-2. It was possible to select cows with different IGF-1 concentrations based upon only 1 blood sample collected in late pregnancy. Concentrations of IGF-1 in IGF-1-low versus IGF-1-high animals (n=7 each) remained significantly different between groups from the day of selection of the animals until d 275 after AI. Second, the differences in total plasma IGF-1 concentration between experimental groups may be attributed to differences in hepatic production of acid labile subunit. The ability of IGFBP-3 to bind IGF-1 declined before calving in all cows. Furthermore, in addition to decreased mRNA expression of growth hormone receptor 1A and IGF-1 relative to calving, serum binding capacities for IGF-1 also decreased. Insulin-like growth factor binding protein 4 mRNA expression was higher in cows with low IGF-1 concentrations; this binding protein inhibits IGF-1 action at the tissue level and therefore may reduce IGF-1 bioavailability. Finally, other endocrine end points (e.g., insulin and thyroid hormones) differed between the 2 groups.

Effects of insulin-like growth factor binding proteins in bone -- a matter of cell and site.

The actions of the insulin-like growth factor (IGF)-system are controlled by six IGF-binding proteins (IGFBPs). The IGFBPs are thought to affect local effects of IGF-I and IGF-II due to higher affinity if compared to IGF-I receptors and due to cell-type specific IGFBP expression patterns. It was found in IGFBP knockout models that the IGFBP family is functionally redundant. Thus, functional analysis of potential effects of IGFBPs is dependent on descriptive studies and models of IGFBP overexposure in vitro and in vivo. In the literature, the role of the IGFBPs for bone growth is highly controversial and, to date, no systematic look has been taken at IGFBPs resolving functional aspects of IGFBPs at levels of cell types and specific locations within bones. Since IGFBPs are thought to represent local modulators of the IGF actions and also exert IGF-independent effects, this approach is particularly reasonable on a physiological level. By sorting the huge number of in part controversial results on IGFBP effects in bone present in the literature for distinct cell types and bone sites it is possible to generate a focused, more specific and a less controversial picture of IGFBP functions in bone.

Browning of subcutaneous fat and higher surface temperature in response to phenotype selection for advanced endurance exercise performance in male DUhTP mice.

For the assessment of genetic or conditional factors of fat cell browning, novel and polygenic animal models are required. Therefore, the long-term selected polygenic mouse line DUhTP originally established in Dummerstorf for high treadmill performance is used. DUhTP mice are characterized by increased fat accumulation in the sedentary condition and elevated fat mobilization during mild voluntary physical activity. In the present study, the phenotype of fat cell browning of subcutaneous fat and a potential effect on oral glucose tolerance, an indicator of metabolic health, were addressed in DUhTP mice. Analysis of peripheral fat pads revealed increased brite (brown-in-white) subcutaneous adipose tissues and in subcutaneous fat from DUhTP mice higher levels of irisin and different markers of fat cell browning like T-box transcription factor (Tbx1), PPARα, and uncoupling protein (UCP1) (P < 0.05) when compared to unselected controls. UCP1 was further increased in subcutaneous fat from DUhTP mice in response to mild exercise (fourfold, P < 0.05). In addition, surface temperature of DUhTP mice was increased when compared to controls indicating a physiological effect of increased UCP1 expression. The present study suggests that DUhTP mice exhibit different markers of mitochondrial biogenesis and fat browning without external stimuli. At an age of 43 days, sedentary DUhTP mice have improved metabolic health as judged from lower levels of blood glucose after an oral glucose tolerance test. Consequently, the non-inbred mouse model DUhTP represents a novel model for the identification of fat cell browning mechanisms in white adipose tissues.

Phenotype analysis of male transgenic mice overexpressing mutant IGFBP-2 lacking the Cardin-Weintraub sequence motif: Reduced expression of synaptic markers and myelin basic protein in the brain and a lower degree of anxiety-like behaviour.

Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (p<0.05). In tissue lysates from brain hemispheres of 12-21day old male mice, protein levels of the GTPase dynamin-I were significantly reduced (p<0.01). Weight reductions were also found in distinct brain regions in two different age groups (12 and 80weeks). In the younger group, impaired weights were observed in the hippocampus (-34%; p<0.001), cerebellum (-25%; p<0.0001), olfactory bulb (-31%; p<0.05) and prefrontal cortex (-29%; p<0.05). At an age of 12weeks expression of myelin basic protein was reduced (p<0.01) in H1d-BP-2 mice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; p<0.001) and hippocampus (-31; p<0.05). When mice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1.

Src stimulates insulin-like growth factor I (IGF-I)-dependent cell proliferation by increasing IGF-I receptor number in human pancreatic carcinoma cells.

We examined the potential function of Src in human pancreatic carcinoma. Overexpression of kinase-activated SrcY527F resulted in a significant increase of insulin-like growth factor I (IGF-I)-dependent cell proliferation in the cell line PANC-1. Western blotting and competition binding studies demonstrated 2.3 +/- 0.2-fold increase in IGF-I receptor expression and 2.8 +/- 0.4-fold increase in IGF-I-specific binding sites/cell. SrcY527F transfection alone did not change receptor affinity or basal receptor tyrosine phosphorylation, whereas IGF-I-stimulated receptor phosphorylation was increased by 2.1 +/- 0.5-fold. IGF-I mRNA expression and protein secretion did not change to exclude autocrine activation. We conclude that Src stimulates IGF-I-dependent proliferation of PANC-1 cells by increasing the number of IGF-I receptors/cell.

Insulin-like growth factor-I is an autocrine regulator of chromogranin A secretion and growth in human neuroendocrine tumor cells.

Carcinoid tumors are predominantly found in the gastrointestinal tract and are characterized by hypersecretion of various substances, including bioamines and neuropeptides, leading to functional tumor disease. Here, we demonstrate that human BON carcinoid tumor cells express functionally active insulin-like growth factor-I (IGF-I) receptors and secrete IGF-I, suggesting an autocrine action of this growth factor. The IGF-I receptor was functionally active. IGF-I stimulated phosphatidylinositol 3-kinase (PI3-kinase), p70 S6 kinase (p70s6k), and extracellular signal-regulated kinase 2 activity in BON cells. Furthermore, immunoneutralization of endogenously released IGF-I markedly reduced the high basal activity of p70s6k and extracellular signal-regulated kinase 2 in serum-starved BON cells. Exogenously added IGF-I induced a marked increase in chromogranin A secretion, a marker protein for neuroendocrine secretion, by a process that was largely dependent on PI3-kinase activity. In addition, immunoneutralization of endogenously released IGF-I markedly reduced basal chromogranin A release by BON cells. Thus, the autocrine IGF-I loop regulates basal neuroendocrine secretion in BON cells. Next, we investigated the role of IGF-I as a growth promoting agent for BON cells. Our data demonstrate that IGF-I stimulates anchorage-dependent and anchorage-independent growth of BON cells by a pathway that involves PI3-kinase, mammalian target of rapamycin/p70s6k, and mitogen-activated protein kinase kinase 1 activity. Interestingly, mitogen-activated protein kinase kinase 1 activity was less important for anchorage-independent growth of BON cells. Endogenously released IGF-I was found to be largely responsible for autonomous growth of BON cells in serum-free medium and for the constitutive expression of cyclin D1 in these cells. In conclusion, IGF-I is a major autocrine regulator of neuroendocrine secretion and growth of human BON neuroendocrine tumor cells. Because our data also demonstrate that a significant proportion of neuroendocrine tumors express the IGF-I receptor and its ligand, interference with this pathway could be useful in the treatment of hypersecretion syndromes and growth of human neuroendocrine tumors.

Overexpression of insulin-like growth factor-binding protein-2 results in increased tumorigenic potential in Y-1 adrenocortical tumor cells.

Increased concentrations of insulin-like growth factor-binding protein-2 (IGFBP-2) have been observed in human malignancies including adrenocortical carcinomas. To elucidate the functional consequences of IGFBP-2 overexpression, we have stably transfected the cDNA of murine IGFBP-2 in mouse adrenocortical tumor cells (Y-1). Long-term overexpression of IGFBP-2 was associated with significant morphological alterations, enhanced cell proliferation, and increased cloning efficiency as compared with mock transfected control cells. The enhanced proliferation of IGFBP-2 secreting clones was independent of exogenous insulin-like growth factors (IGFs). These data suggest that elevated levels of IGFBP-2 may contribute to the highly malignant phenotype of adrenocortical cancer by a thus far unknown, presumably IGF-independent, mechanism.

Perturbation of C6 glial cells by acetate leads to modulation of [125I]IGF-II binding to the IGF-II/M6P receptor: implications for ligand internalization studies.

Acid washes are used as an experimental tool to differentiate between cell-surface bound and internalized radioligands. We have observed that washes with acid buffers containing 100 mM acetate can modulate [125I]IGF-II binding to rat C6 glial cells in an unexpected manner: when cells in monolayer culture were prewashed with phosphate buffered saline (pH 7.3) (PBS), [125I]IGF-II binding was characteristic of the IGF-II/mannose-6-phosphate (M6P) receptor. Importantly, IgG 3637, which is purified from an antiserum directed against the rat IGF-II/M6P receptor, blocked binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity crosslinking studies using DSS as the crosslinking agent and Western blotting experiments using antiserum 3637 confirmed the presence of the IGF-II/M6P receptor in C6 glial cells. Prewashes of C6 cell monolayers with acid buffers (pH 4-4.5) which contained 100 mM sodium acetate and which have been used in internalization studies reduced [125I]IGF-II binding by 40-60%. Affinity crosslinking studies using C6 cells showed that the formation of the 250 kDa radioligand-receptor complex was not prohibited by IgG 3637 after acid washes with buffers containing high acetate concentrations, while acid washes with buffers containing no acetate did not cause a loss in the blocking ability of IgG 3637. However, acid washes with 100 mM acetate did not alter the recognition of IGF-II/M6P receptors by IgG 3637 in Western blotting experiments. In addition, in a subset of experiments acid prewashes with acetate also decreased binding of [125I]IGF-I to the IGF-I receptor by 20%. We conclude that acid washes with acetate buffers lead to decreased [125I]IGF-I and [125I]IGF-II binding. In addition, the capability of anti-receptor IgG to block radioligand binding to the IGF-II/M6P receptor also declines. We hypothesize that alteration of ligand binding might be partially caused by perturbation of the cell membrane and hence a conformational change in IGF receptors. These data imply that the use of acetate buffers in acid wash experiments in ligand internalization studies--particularly in studies involving the IGF-II/M6P receptor--should be avoided.

Opposite regulation of IGF-I and IGF-I receptor mRNA and concomitant changes of GH receptor and IGF-II/M6P receptor mRNA in human IM-9 lymphoblasts.

Human IM-9 lymphoblasts synthesize IGF-I and express IGF-I receptors, IGF-II/M6P receptors and GH receptors. We have studied the regulation of mRNA expression of IGF-I, IGF-I receptors, IGF-II/M6P receptors and GH receptors in IM-9 cells upon serum-withdrawal and re-addition of serum. IM-9 cells were cultured in RPMI-1640 medium with or without serum for various periods of time. RNA was prepared using guanidinium thiocyanate and CsCl. Antisense riboprobes for human IGF-I, IGF-I receptor, IGF-II/M6P receptor, GH receptor and for comparison for human beta-actin were synthesized and labeled with 32P. Protected fragments of 379 bases and of 420 and 350 bases with the IGF-I receptor and with the IGF-I probe respectively and protected fragments of 670 bases and of 51 and 121 bases with the GH receptor and with the beta-actin probe were detected. For the human IGF-II/M6P receptor probe protected fragments of 260 bases were visualized in RNA samples. The amount of mRNA present in each lane (10 microgram total RNA) was determined by computed densitometry. The amount of IGF-mRNA expressed by IM-9 cells decreased rapidly (within two hours) and dramatically (more than 120%) after the withdrawal of serum and increased significantly (220%) after the re-addition of serum. This increase of IGF-I mRNA preceded the increase in cell number that was seen after 48 h of medium change. Conversely, the expression of IGF-I receptor mRNA and beta-actin mRNA increased by more than 250% after the withdrawal of serum within 2 and 8 h respectively, while GH receptor mRNA fell within 2-4 h. The expression of IGF-II/M6P receptor mRNA continued to increase throughout the duration of the cell culture experiment. We conclude that IGF-I and IGF-I receptor mRNAs are regulated in an opposite direction in serum-deprived IM-9 lymphoblasts. In addition, GH receptor mRNA expression parallels IGF-I mRNA expression.

Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation, migration, and invasion.

IGF binding proteins (IGFBPs) modulate IGF cellular bioavailability and may directly regulate tumor growth and invasion. We have previously shown that IGFBP-2 binds and localizes IGF-I to the pericellular matrix and have provided some evidence suggesting that the heparin binding domain (HBD) or the arginine-glycine-aspartic acid (RGD) integrin binding motif may be involved in these interactions. However, the precise mechanisms involved remain to be elucidated. We therefore mutated the HBD or RGD sequence of IGFBP-2 and investigated consequent effects on extracellular matrix (ECM) binding, IGF-induced proliferation, and migration of neuroblastoma cells. IGFBP-2 and its arginine-glycine-glutamic acid (RGE) mutant similarly bound ECM components, whereas binding of mutant HBD-IGFBP-2 to each of the ECM substrates was markedly reduced by 70-80% (P < 0.05). IGF-I (100 ng/ml) increased incorporation of 3H-thymidine in neuroblastoma SK-N-SHEP cells by approximately 30%, an effect blunted by exogenously added native or either mutant IGFBP-2. Overexpression of IGFBP-2 and its RGE mutant potently promoted SHEP cell proliferation (5-fold), whereas SHEP cell proliferation was negligible when HBD-IGFBP-2 was overexpressed. Addition or overexpression of IGFBP-2 and its RGE mutant potently (P < 0.05) enhanced SHEP cell migration/invasion through the ECM. However, overexpression of the HBD-IGFBP-2 mutant potently inhibited (50-60%) SHEP cell invasion through ECM. Thus, IGFBP-2, which binds to the ECM, enhances proliferation and metastatic behavior of neuroblastoma cells, functions that directly or indirectly use the HBD but not the integrin binding sequence. Our novel findings thus point to a key role for the HBD of IGFBP-2 in the control and regulation of neuroblastoma growth and invasion.

Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain.

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) has been shown to inhibit IGF-dependent cell proliferation in a number of in vitro studies. However, no in vivo model of IGFBP-2 overexpression has been established so far. Therefore, we have generated transgenic mice, in which expression of a mouse IGFBP-2 complementary DNA is controlled by the cytomegalovirus (CMV) promoter. In two independent transgenic strains, transgene expression was highest in pancreas and stomach, followed by skeletal muscle, heart, colon, spleen, adipose tissue, brain, and kidney. Within the pancreas, IGFBP-2 expression was found in the islets but not in the exocrine part. Serum IGFBP-2 levels of CMV-IGFBP-2 transgenic mice were about 3-fold (P < 0.05) increased, compared with controls, whereas serum levels of IGF-I and IGF-II were unaffected by IGFBP-2 overexpression. Fasted serum glucose and fasted insulin levels were slightly reduced in transgenic mice, compared with controls. Postprandial serum glucose insulin levels were not affected by the genotype. At days later than 23, body weights of transgenic mice were significantly (P < 0.05) reduced in both sexes, compared with nontransgenic littermates. This reduction in body weight was mainly attributable to significantly (P < 0.05) lower carcass weights of CMV-IGFBP-2 transgenic vs. control mice. In contrast, absolute organ weights were not (or only as a tendency) reduced, except for the weight of the spleen, which was significantly (P < 0.05) lower in male transgenic than in control mice. Our data suggest that IGFBP-2 represents a negative regulator of postnatal growth in mice, potentially by reducing the bioavailability of IGF-I.

Growth inhibition in giant growth hormone transgenic mice by overexpression of insulin-like growth factor-binding protein-2.

To clarify the role of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) in postnatal growth regulation, we crossed hemizygous CMV-IGFBP-2 transgenic mice with hemizygous PEPCK-bGH transgenic mice, which are characterized by serum GH levels in the range of 2 microgram/ml. Four genetic groups were obtained: animals carrying both transgenes (GB), the GH (G) or the IGFBP-2 transgene (B), and nontransgenic controls (C). Male offspring were analyzed for serum levels of IGF-I, for serum and tissue levels of IGFBP-2, and for body and organ growth. Serum IGF-I levels were 2- to 3-fold increased (P < 0.001) in the GH-overexpressing groups, with no difference between G and GB mice. Serum IGFBP-2 levels were 4- to 9-fold (P < 0.001) increased both in B and GB vs. C and G mice. Western immunoblot analysis did not reveal differences in tissue IGFBP-2 levels between B and GB mice. IGFBP-2 levels were highest in pancreas, followed by skeletal muscle, heart, kidney, brain, skin, and spleen. No elevation of IGFBP-2 was found in the liver. Body weight gain of G and GB mice was significantly increased vs. C and B mice, resulting in almost 2-fold increased body weights at the age of 15 weeks. However, there was a significant reduction in body weight of GB vs. G mice (17%; P < 0.001) and of B vs. C mice (13%; P < 0.05). This was primarily caused by a marked reduction of carcass weight (GB vs. G, 27%; B vs. C, 21%; P < 0.001). Measurements of nose-rump-length, organ (brain, heart, spleen, liver, pancreas, kidney), and tissue weights (skin, carcass, abdominal fat) in 5- and 15-week-old mice revealed several indications that the growth-inhibiting effect of IGFBP-2 overexpression was more marked in high-GH/IGF-I mice: 1) At 5 weeks of age, GB mice displayed a significant reduction of all growth parameters except for the weight of abdominal fat, when compared with G mice, whereas only brain weight was significantly reduced in B vs. C mice. 2) In 15-week-old animals, a significant reduction in all growth parameters, except for spleen and abdominal fat weights, was seen in GB vs. G mice, whereas only nose-rump-length and the weights of carcass and brain were significantly reduced in B vs. C mice. Our study demonstrates, for the first time, the potential of IGFBP-2 to inhibit GH-stimulated growth in giant transgenic mice, providing further evidence for an inhibitory effect of this IGFBP in vivo.