Glucagon orchestrates stress-induced hyperglycaemia.

Hyperglycaemia is commonly observed on admission and during hospitalization for medical illness, traumatic injury, burn and surgical intervention. This transient hyperglycaemia is referred to as stress-induced hyperglycaemia (SIH) and frequently occurs in individuals without a history of diabetes. SIH has many of the same underlying hormonal disturbances as diabetes mellitus, specifically absolute or relative insulin deficiency and glucagon excess. SIH has the added features of elevated blood levels of catecholamines and cortisol, which are not typically present in people with diabetes who are not acutely ill. The seriousness of SIH is highlighted by its greater morbidity and mortality rates compared with those of hospitalized patients with normal glucose levels, and this increased risk is particularly high in those without pre-existing diabetes. Insulin is the treatment standard for SIH, but new therapies that reduce glucose variability and hypoglycaemia are desired. In the present review, we focus on the key role of glucagon in SIH and discuss the potential use of glucagon receptor blockers and glucagon-like peptide-1 receptor agonists in SIH to achieve target glucose control.

A clinical trial assessing the safety and efficacy of the CB1R inverse agonist taranabant in obese and overweight patients: low-dose study.

OBJECTIVE: To evaluate the weight loss efficacy, safety and tolerability of taranabant, a CB1R inverse agonist, in obese and overweight patients. DESIGN: Multicenter, double-blind, randomized, placebo-controlled study. SUBJECTS: Patients >or=18 years old, BMI 27-43 kg m(-2), were randomized to placebo (n=209) or taranabant 0.5 mg (n=207), 1 mg (n=208) or 2 mg given orally once daily (n=417) for 52 weeks. MEASUREMENTS: Key efficacy measurements included body weight (BW), waist circumference (WC), lipid endpoints and glycemic endpoints. RESULTS: Based on a last observation carried forward analysis of the all-patients-treated population, mean change in BW for taranabant 0.5, 1, and 2 mg and placebo was -5.4, -5.3, -6.7 and -1.7 kg, respectively (P<0.001 for all doses vs placebo). The proportions of patients who lost at least 5 and 10% of their baseline BW at week 52 were significantly higher for all taranabant doses vs placebo (P<0.001 for all doses). Reductions in WC, percentage of body fat, and triglycerides were significant for taranabant 2 mg and in triglycerides for taranabant 1 mg vs placebo. There was no effect of taranabant vs placebo on other lipid or glucose-related endpoints. Incidences of adverse experiences classified in the gastrointestinal (diarrhea and nausea), nervous system (dizziness/dizziness postural), psychiatric-related (irritability and anger/aggression) and vascular (flushing/hot flush) organ systems were higher and statistically significant in the taranabant 2-mg group compared with the placebo group. Irritability was higher and statistically significant in all taranabant groups compared with the placebo group. CONCLUSION: All three doses of taranabant-induced clinically meaningful and statistically significant weight loss. Incidences of adverse experiences in organ systems known to express CB1R were higher in taranabant groups.

How much may I eat? Calorie estimates based upon energy expenditure prediction equations.

How much may I eat? Most healthcare workers, when asked this question, have insufficient knowledge to educate their patients on a healthy energy intake level. In this review we examine the available methods for estimating adult energy requirements with a focus on the newly developed National Academy of Sciences/Institute of Medicine (NAS/IOM) doubly-labelled water total energy expenditure (TEE) prediction equations. An overview is first provided of the traditional factorial method of estimating energy requirements. We then extend this overview by exploring the development of the NAS/IOM TEE prediction models and their role in estimating energy requirements as a function of sex, age, weight, height and physical activity level. The NAS/IOM prediction models were developed for evaluating group energy requirements, although the formulas can be applied in individual 'example' patients for educational purposes. Potential limitations and interpretation issues of both the factorial and NAS/IOM methods are examined. This information should provide healthcare professionals with the tools and understanding to appropriately answer the question, 'How much may I eat?'

Differential expression of signal transducers and activators of transcription during human adipogenesis.

Signal Transducers and Activators of Transcription (STATs) display unique expression patterns upon induction of differentiation of murine 3T3-L1 preadipocytes into adipocytes. During differentiation, expression of STAT1 and STAT5 increase, while STAT3 and STAT6 remain relatively unchanged. Here, we determined whether human subcutaneous preadipocytes expressed STATs and if the pattern of expression changed during adipogenesis. We found by Western blot analysis that freshly isolated preadipocytes expressed STAT1, STAT3, STAT5, and STAT6, but not STAT2 and STAT4. Induction of preadipocyte differentiation with 1-methyl-3-isobutylxanthine, dexamethasone, insulin, and BRL49653 decreased expression of STAT1, and increased expression of STAT3 and STAT5. STAT6 expression did not change during adipogenesis. Changes in expression of CCAAT/enhancer binding protein beta (C/EBPbeta), C/EBPdelta, C/EBPalpha, and peroxisome proliferator-activated receptor gamma were similar to murine cell lines. These results suggest that unlike the traditional adipogenic transcription factors, unique differences exist in STAT expression patterns between murine and human adipose cells.

Differential effects of flavonoids on 3T3-L1 adipogenesis and lipolysis.

Flavonoids, polyphenolic compounds that exist widely in plants, inhibit cell proliferation and increase cell differentiation in many cancerous and noncancerous cell lines. Because terminal differentiation of preadipocytes to adipocytes depends on proliferation of both pre- and postconfluent preadipocytes, we predicted that flavonoids would inhibit adipogenesis in the 3T3-L1 preadipocyte cell line. The flavonoids genistein and naringenin inhibited proliferation of preconfluent preadipocytes in a time- and dose-dependent manner. When added to 2-day postconfluent preadipocytes at the induction of differentiation, genistein inhibited mitotic clonal expansion, triglyceride accumulation, and peroxisome proliferator-activated receptor-gamma expression, but naringenin had no effect. The antiadipogenic effect of genistein was not due to inhibition of insulin receptor subtrate-1 tyrosine phosphorylation. When added 3 days after induction of differentiation, neither flavonoid inhibited differentiation. In fully differentiated adipocytes, genistein increased basal and epinephrine-induced lipolysis, but naringenin had no significant effects. These data demonstrate that genistein and naringenin, despite structural similarity, have differential effects on adipogenesis and adipocyte lipid metabolism.

Activation of signal transducer and activator of transcription-3 during proliferative phases of 3T3-L1 adipogenesis.

Signal transducer and activator of transcription-3 (STAT3) is abundantly expressed in preadipocytes and adipocytes, but little is known about its activation status or functional role during adipogenesis. In this report we investigate STAT3 activation in 3T3-L1 preadipocytes before and after differentiation into adipocytes. STAT3 was highly tyrosine phosphorylated and bound to DNA in proliferating preadipocytes, but not in growth-arrested preadipocytes or adipocytes. In growth-arrested confluent preadipocytes, induction of differentiation with methylisobutylxanthine, dexamethasone, and high dose insulin led to a delayed, but prolonged (3-day), increase in STAT3 tyrosine phosphorylation. This increase in STAT3 phosphorylation coincided temporally with postconfluent preadipocyte mitotic clonal expansion. Insulin and methylisobutylxanthine alone, but not dexamethasone, induced STAT3 tyrosine phosphorylation in postconfluent cells. Diminution of endogenous STAT3 expression by antisense morpholino oligonucleotides significantly decreased preconfluent preadipocyte proliferation. Collectively, these findings suggest a regulatory role for STAT3 during the proliferative phases of adipogenesis.

Interleukin-4 mediates STAT6 activation in 3T3-L1 preadipocytes but not adipocytes.

STAT6 is abundantly expressed in 3T3-L1 preadipocytes and adipocytes but activating ligands are not well defined. In this report, we provide evidence that interleukin 4 (IL-4) induced JAK2-mediated STAT6 tyrosine phosphorylation and DNA binding in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. Loss of IL-4-mediated STAT6 tyrosine phosphorylation occurred 2 days after preadipocytes were induced to differentiate into adipocytes but when cells remained phenotypically preadipocytes. 3T3-L1 adipocytes were still responsive to IL-4 through tyrosine phosphorylation of other cellular proteins. We conclude that IL-4 signals through STAT6 in 3T3-L1 preadipocytes but not in 3T3-L1 adipocytes. This differentiation-dependent loss of STAT6 activation may be critical for distinct biological effects of IL-4 in 3T3-L1 preadipocytes and adipocytes.

Orlistat for the long-term treatment of obesity.

Orlistat, a potent gastrointestinal lipase inhibitor, is a member of a new class of drugs designed for the long-term treatment of obesity. When given with a fat-containing meal, orlistat reduces dietary fat absorption by approximately 30%, which equates to a decrease in caloric absorption of approximately 200 kilocalories per day. A 2-year European study found a mean decrease in body weight of 10.2% (10.3 kg) in the orlistat group compared to 6.1% (6.1 kg) in the placebo group at 1 year. Additionally, 9.3% of the orlistat group versus 2.1% of the placebo group lost >20% of their initial weight. Serum lipids and diabetes control are also improved by orlistat. Related to orlistat's mechanism of action, side effects include oily spotting, flatulence and frequent loose stools, but not frank diarrhea or intestinal malabsorption. Vitamin D and beta-carotene levels decreased, but remained within the normal range. In summary, orlistat is the first example of a new class of antiobesity drugs that enhances weight loss and weight maintenance by interfering with dietary fat absorption. Orlistat has tolerable gastrointestinal side effects and no major drug toxicity. Orlistat is a viable adjunct to lifestyle interventions used in the long-term management of obesity.

Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C.

p130Cas is a signaling molecule that was initially found to be tyrosine-phosphorylated in v-Crk and v-Src transformed cells. We characterized the regulation of p130Cas tyrosine phosphorylation in vascular smooth muscle cells by angiotensin II (Ang II). This ligand induced a transient increase in p130Cas tyrosine phosphorylation, which was sensitive to the actin polymerization inhibitor cytochalasin D and to the intracellular Ca2+ chelator BAPTA-AM but not the Ca2+ channel blocker verapamil. The Ang II-induced tyrosine phosphorylation of p130Cas was also dependent on an active Src family tyrosine kinase, since it could be blocked by the Src kinase inhibitors geldanamycin and PP1. Ang II treatment resulted in the ability of p130Cas to bind at least 11 different phosphate-containing proteins. Analysis of these proteins revealed that protein kinase Calpha and the cell adhesion signaling molecule pp120 formed temporal associations with p130Cas in response to Ang II. c-Src was found to associate with p130Cas in a manner that was independent of Ang II treatment. Inhibition of protein kinase C by either calphostin C or phorbol 12-myristate 13-acetate downregulation inhibited the Ang II-induced tyrosine phosphorylation of p130Cas. These results are the first to demonstrate that the tyrosine phosphorylation of p130Cas by Ang II is transduced by the Src, intracellular Ca2+, protein kinase C signaling pathway.

Angiotensin II-induced association of phospholipase Cgamma1 with the G-protein-coupled AT1 receptor.

An early event in signaling by the G-protein-coupled angiotensin II (Ang II) AT1 receptor in vascular smooth muscle cells is the tyrosine phosphorylation and activation of phospholipase Cgamma1 (PLCgamma1). In the present study, we show that stimulation of this event by Ang II in vascular smooth muscle cells is accompanied by binding of PLCgamma1 to the AT1 receptor in an Ang II- and tyrosine phophorylation-dependent manner. The PLCgamma1-AT1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT1 receptor and binding of the phosphorylated receptor by the most C-terminal of two Src homology 2 domains in PLCgamma1. PLCgamma1 thus binds to the same site in the receptor previously identified for binding by the SHP-2 phosphotyrosine phosphatase.JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the AT1 receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. These data demonstrate that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors.

Role of Janus kinase/signal transducer and activator of transcription and mitogen-activated protein kinase cascades in angiotensin II- and platelet-derived growth factor-induced vascular smooth muscle cell proliferation.

In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors.

Role of intracellular calcium in the angiotensin II-mediated tyrosine phosphorylation and dephosphorylation of PLC-gamma 1.

Angiotensin II induces the rapid temporal tyrosine phosphorylation and activation of phospholipase C-gamma 1 (PLC-gamma 1) and the elevation of intracellular calcium levels. To investigate the relationship of these intracellular signaling events, rat aortic smooth muscle cells were treated with the calcium chelator BAPTA-AM, the calcium channel blocker verapamil, the intracellular calcium antagonist TMB-8, and the calcium ionophore ionomycin. The effects of these agents on PLC-gamma 1 tyrosine phosphorylation were then measured. We found that treatment of these cells with the calcium inhibitors augmented the basal level of PLC-gamma 1 tyrosine phosphorylation, without changing the peak level of tyrosine phosphorylation induced by angiotensin II. The rapid dephosphorylation of PLC-gamma 1 that follows angiotensin II stimulation was prevented by these calcium antagonists. In contrast, angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 was inhibited by ionomycin. These results suggest that the angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 is calcium-independent, while the dephosphorylation is calcium-dependent.

Components of the renin-angiotensin system in adipose tissue: changes with maturation and adipose mass enlargement.

White adipose tissue is a rich source of angiotensinogen protein and mRNA. Studies in clonal cells suggest that angiotensinogen, and its cleavage product, angiotensin II, are involved in preadipocyte differentiation into mature fat cells. No studies have determined whether angiotensinogen is also involved in adipose tissue development in vivo. In this report, we studied male Wistar rats at two stages of development to determine if angiotensinogen protein and mRNA are increased in retroperitoneal fat depots of rapidly growing young, lean, 8-week-old rats compared to 26-week-old rats that are fatter, but are undergoing less rapid adipose tissue growth. We also assessed renin mRNA and angiotensin I-generating activity, since it is less clear whether renin is locally produced in adipose tissue. We found that angiotensin I-generating activity was measurable in adipose tissue and adipocytes, but renin mRNA was undetectable by northern blot analysis. Angiotensinogen mRNA was abundant in adipocytes, but was absent in stromal-vascular cells of adipose tissue. Angiotensinogen content per 10 million fat cells was approximately threefold higher in 8-week-old rats compared to 26-week-old rats (p < .0002). Angiotensinogen mRNA was approximately twofold higher in adipocytes of 8-week-old rats compared to 26-week-old rats. The age-related decline in angiotensinogen protein and mRNA indicates that the local renin-angiotensin system may play an important role in adipose tissue growth, and possibly contribute to the changes in adipose mass and cellularity seen in old senescent rats.

Simplified enzymatic assay of angiotensin-converting enzyme in serum.

We report a simple, enzymatic method for determining angiotensin-converting enzyme (ACE; EC 3.4.15.1) in serum. The proposed method features coupling an established reaction catalyzed by gamma-glutamyltransferase (GGT; EC 2.3.2.2) to the ACE reaction, which releases glycylglycine from the artificial substrate hippuryl-glycyl-glycine. The glycyl-glycine released by the ACE reaction becomes rate-limiting in the GGT reaction, in which it participates as a receptor substrate for a gamma-glutamyl group transferred from a donor substrate, L-gamma-glutamyl-3-carboxy-4-nitroanilide. The reaction releases 3-carboxy-4-nitroaniline, which is monitored spectrophotometrically at 410 nm. The resulting rate of change in absorbance is linearly related to the glycyl-glycine concentration and therefore to the activity of ACE. The linear range of the method extends from ACE values < 50 to at least 1300 U/L of serum. Good precision is indicated by a low CV for replicate analyses (3.6% and 4.6% for within-run and day-to-day assays, respectively, for normal ACE activity, and 3.1% within-run for high ACE activity). Results also correlate well with those of an established colorimetric method (r = 0.978). The major advantages of the method are its procedural simplicity, limited cost, use of readily available reagents, applicability to isoenzyme studies, and adaptability to automation.

Nutrition and somatomedin. XXII: Molecular regulation of insulin-like growth factor-I during fasting and refeeding in rats.

The liver is thought to be the locus of nutritional/hormonal regulation of circulating insulin-like growth factor-I (IGF-I). To probe the basis of nutritional regulation, we examined changes in serum IGF-I, hepatic content of extractable IGF-I immunoreactivity (a high Mr putative precursor) and hepatic IGF-I mRNA during fasting and refeeding in rats. Preliminary studies revealed that the hepatic level of IGF-I mRNA was consistently reduced only after food was withheld for 3 days, so the effects of refeeding were subsequently examined in such animals. After 3 days of fasting, animals lost 30% of their initial weight; weight regain was apparent within 3 h of refeeding ad libitum and, after 48 h, weight was comparable to initial fed levels. Fasting reduced levels of serum and extractable hepatic IGF-I to 19 and 26% of control (fed) values respectively (both P less than 0.005 vs control). There was no change in levels of serum IGF-I over the first 3 h of refeeding, but IGF-I rose above fasted levels at both 9 and 48 h (both P less than 0.005). Extractable hepatic IGF-I rose more slowly and was still below fasted levels after 9 h of refeeding, and modestly, but not significantly, greater than fasted levels after 48 h. The ratio of serum to hepatic IGF-I was decreased compared with control after 3 days of fasting, but increased after 3 and 9 h of refeeding (P less than 0.02 at 9 h). Northern blot analysis of total hepatic RNA revealed four species of IGF-I mRNA 0.8-1.1, 2.0, 4.0 and 7.5 kb in size. Each mRNA species fell to 15-28% of control levels after 3 days of fasting (all P less than 0.001). There was a prompt increase in each transcript after 3 h of refeeding, and all values were significantly (P less than 0.05) greater than fasted levels at 9 h but, at 48 h, most species were still below control levels. Levels of mRNA for the cytoskeletal proteins beta-actin and cyclophilin also fell with fasting, but were restored more rapidly than IGF-I mRNA, to or above control levels after 3 h of refeeding. The observation that IGF-I expression was decreased at 3 h when beta-actin and cyclophilin were normalized suggests specificity of regulation. Despite the temporal incongruity between IGF-I mRNA and serum and hepatic IGF-I, there were highly significant correlations (all P less than 0.002) between each pair of parameters.(ABSTRACT TRUNCATED AT 400 WORDS)

Nutrition and somatomedin. XXIII. Molecular regulation of IGF-I by amino acid availability in cultured hepatocytes.

The poor growth associated with protein-calorie malnutrition occurs despite circulating growth hormone levels that are normal or elevated and is thought to be mediated partly by blunted generation of insulinlike growth factor I (IGF-I) in the liver. To explore underlying mechanisms, we asked whether altered availability of amino acids could regulate hepatic IGF-I release independent of the contributions of regulatory hormones. Normal rat hepatocytes were isolated by collagenase digestion and maintained in serum-free medium with fixed concentrations of insulin and dexamethasone. Levels of immunoassayable albumin and IGF-I accumulation in daily changes of medium were sustained for 3-5 days, and all studies were performed within this period. Cellular viability and content of DNA were unaffected by deprivation of the essential amino acids lysine or tryptophan and the nonessential amino acids cysteine and/or cystine. However, deletion of tryptophan or lysine from the culture medium led to 63 and 76% declines in IGF-I release, respectively (both P less than 0.001 vs. complete medium), although omission of cysteine or cysteine plus cystine produced no significant change. Over 5 days of culture, release of albumin was maintained in complete medium, but omission of tryptophan depressed albumin release over days 2-5 (P less than 0.001). In complete medium, IGF-I release rose for 3 days and then declined. In tryptophan-deficient medium, IGF-I levels were comparable to control values after 24 h but did not rise at 48 h and then fell rapidly after 72 h in culture, with values significantly below levels in complete medium (all P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation and action of insulin-like growth factors at the cellular level.

Present understanding of IGF-1 as a growth factor mediating integration of nutritional-hormonal interactions indicates that IGF-1 acts in both an endocrine mode on distant targets and an autocrine-paracrine mode on local targets. In the liver, the combined presence of GH, insulin, and critical metabolic fuels such as essential amino acids results in increased levels of IGF-1 messenger RNA, increased production of a high-MW IGF-1 precursor, and increased release of IGF-1 into the circulation, permitting action on distant target tissues bearing specific receptors for IGF-1. The net effect is distant amplification of anabolic hormone action via IGF-1 acting in an endocrine mode. In extrahepatic tissues, both 'general' anabolic hormones (insulin and GH) as well as 'specific' hormones (e.g. gonadotropins) acting on a wide variety of targets (including fibroblasts and chondrocytes as well as granulosa and Leydig cells) promote both local secretion of IGF-1 and an increase in IGF-1 receptors. Local actions of IGF-1 then result in a secondary increase in both hormone receptors and hormone responses. The net effect is local amplification of hormone action via IGF-1 acting as a growth factor in an autocrine-paracrine mode.