Impaired awareness of hypoglycaemia in women with type 1 diabetes in pregnancy: Hypoglycaemia fear, glycaemic and pregnancy outcomes.

AIMS: To examine maternal fear of hypoglycaemia, glycaemia and pregnancy outcomes in women with impaired and normal awareness of hypoglycaemia. METHODS: A pre-planned sub-study of 214 pregnant women with type 1 diabetes who participated in the CONCEPTT trial. Participants completed hypoglycaemia fear surveys (HFS-II) at baseline. Logistic regression and Poisson regression analyses were used to obtain an adjusted estimate for the rate ratio relating awareness to the number of severe hypoglycaemic episodes, and for several neonatal outcomes in relation to the total HFS-II score. The role of continuous glucose monitoring (CGM) use was examined. RESULTS: Overall, 30% of participants reported impaired awareness of hypoglycaemia (n = 64). Women with impaired awareness of hypoglycaemia had more episodes of severe hypoglycaemia (mean 0.44 vs. 0.08, p < 0.001) (12-34 weeks gestation) and scored higher on the HFS-II scale (43.7 vs. 36.0, p 0.008), indicating more fear of hypoglycaemia. They spent more time below range (CGM <3.5 mmol/L) and exhibited more glycaemic variability at 12 weeks gestation. Higher overall HFS-II scores were associated with a higher risk of maternal severe hypoglycaemia episodes (Rate Ratio 1.78, 95% CI 1.39-2.27). Women with impaired awareness of hypoglycaemia had less maternal weight gain but there were no differences in neonatal outcomes between women with impaired awareness of hypoglycaemia and normal hypoglycaemia awareness. Higher HFS-II scores were associated with more nephropathy (Odds Ratio 1.91, 95% CI 1.06-3.4). CGM use after 12 weeks was not associated with the number of episodes of severe hypoglycaemia (RR 0.75, 95% CI 0.49-1.15; p = 0.18). CONCLUSIONS: In pregnant women with type 1 diabetes, impaired awareness of hypoglycaemia is associated with more maternal severe hypoglycaemia episodes and more fear of hypoglycaemia. Having impaired awareness of hypoglycaemia and/or fear of hypoglycaemia should alert clinicians to this increased risk. Reassuringly, there was no increase in adverse neonatal outcomes.

ErbB activity links the glucagon-like peptide-2 receptor to refeeding-induced adaptation in the murine small bowel.

BACKGROUND & AIMS: The small bowel mucosa is sensitive to nutrients and undergoes rapid adaptation to nutrient deprivation and refeeding through changes in apoptosis and cell proliferation, respectively. Although glucagon-like peptide-2 (GLP-2) exerts trophic effects on the gut and levels increase with refeeding, mechanisms linking GLP-2 to mucosal adaptation to refeeding remain unclear. METHODS: Fasting and refeeding were studied in wild-type (WT) and Glp2r(-/-) mice and in WT mice treated with the pan ErbB inhibitor CI-1033. Experimental end points included intestinal weights, histomorphometry, gene and protein expression, and crypt cell proliferation. RESULTS: Fasting was associated with significant reductions in small bowel mass, decreased crypt plus villus height, and reduced crypt cell proliferation. Refeeding increased plasma levels of GLP-2, reversed small bowel atrophy, increased villus height and cell number, and stimulated jejunal crypt cell proliferation. In contrast, refeeding failed to increase small bowel weight, crypt cell proliferation, or villus cell number in Glp2r(-/-) mice. Levels of mRNA transcripts for egf, kgf, and igfr were lower in fasted Glp2r(-/-) mice. Epidermal growth factor but not insulin-like growth factor-1 restored the intestinal adaptive response to refeeding in Glp2r(-/-) mice. Furthermore, CI-1033 prevented adaptive crypt cell proliferation, Akt activation, and induction of ErbB ligand gene expression after refeeding. Up-regulation of ErbB ligand expression and intestinal Akt phosphorylation were significantly diminished in refed Glp2r(-/-) mice. CONCLUSIONS: These findings identify Glp2r and ErbB pathways as essential components of the signaling network regulating the adaptive mucosal response to refeeding in the mouse intestine.

Glucagon-like peptide-2 receptor modulates islet adaptation to metabolic stress in the ob/ob mouse.

BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is a gut hormone that increases gut growth, reduces mucosal cell death, and augments mesenteric blood flow and nutrient absorption. Exogenous GLP-2(1-33) also stimulates glucagon secretion and enhances gut barrier function with implications for susceptibility to systemic inflammation and subsequent metabolic dysregulation. We examined the importance of GLP-2 receptor (GLP-2R) signaling for glucose homeostasis in multiple models of metabolic stress, diabetes, and obesity. METHODS: Body weight, islet function, glucose tolerance, and islet histology were studied in wild-type, high-fat fed, lean diabetic, Glp2r(-/-) and ob/ob:Glp2r(-/-) mice. RESULTS: GLP-2 did not stimulate glucagon secretion from isolated pancreatic islets in vitro, and exogenous GLP-2 had no effect on the glucagon response to insulin-induced hypoglycemia in vivo. Glp2r(-/-) mice exhibit no change in glycemia, and plasma glucagon levels were similar in Glp2r(-/-) and Glp2r(+/+) mice after hypoglycemia or after oral or intraperitoneal glucose challenge. Moreover, glucose homeostasis was comparable in Glp2r(-/-) and Glp2r(+/+) mice fed a high-fat diet for 5 months or after induction of streptozotocin-induced diabetes. In contrast, loss of the GLP-2R leads to increased glucagon secretion and alpha-cell mass, impaired intraperitoneal glucose tolerance and hyperglycemia, reduced beta-cell mass, and decreased islet proliferation in ob/ob:Glp2r(-/-) mice. CONCLUSIONS: Our results show that, although the GLP-2R is not critical for the stimulation or suppression of glucagon secretion or glucose homeostasis in normal or lean diabetic mice, elimination of GLP-2R signaling in obese mice impairs the normal islet adaptive response required to maintain glucose homeostasis.

Glucagon-like peptide-2 increases intestinal lipid absorption and chylomicron production via CD36.

BACKGROUND & AIMS: Excessive postprandial lipemia is a prevalent condition that results from intestinal oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins. Glucagon-like peptide-2 (GLP-2) is a gastrointestinal-derived intestinotropic hormone that links nutrient absorption to intestinal structure and function. We investigated the effects of GLP-2 on intestinal lipid absorption and lipoprotein production. METHODS: Intestinal lipid absorption and chylomicron production were quantified in hamsters, wild-type mice, and Cd36(-/-) mice infused with exogenous GLP-2. Newly synthesized apoB48 was metabolically labelled in primary hamster jejunal fragments. Fatty acid absorption was measured, and putative fatty acid transporters were assessed by immunoblotting. RESULTS: Human GLP-2 increased secretion of the triglyceride (TG)-rich lipoprotein (TRL)-apoB48 following oral administration of olive oil to hamsters; TRL and cholesterol mass each increased 3-fold. Fast protein liquid chromatography profiling indicated that GLP-2 stimulated secretion of chylomicron/very low-density lipoprotein-sized particles. Moreover, GLP-2 directly stimulated apoB48 secretion in jejunal fragments cultured ex vivo, increased expression of fully glycosylated cluster of differentiation 36/fatty acid translocase (CD36), and induced intestinal absorption of [(3)H]triolein. The ability of GLP-2 to increase intestinal lipoprotein production was lost in Cd36(-/-) mice. CONCLUSIONS: GLP-2 stimulates intestinal apoB48-containing lipoprotein secretion, possibly through increased lipid uptake, via a pathway that requires CD36. These findings suggest that GLP-2 represents a nutrient-dependent signal that regulates intestinal lipid absorption and the assembly and secretion of TRLs from intestinal enterocytes.

Enteroendocrine-derived glucagon-like peptide-2 controls intestinal amino acid transport.

OBJECTIVE: Glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 from gut endocrine cells, and both peptides act as growth factors to expand the surface area of the mucosal epithelium. Notably, GLP-2 also enhances glucose and lipid transport in enterocytes; however, its actions on control of amino acid (AA) transport remain unclear. Here we examined the mechanisms linking gain and loss of GLP-2 receptor (GLP-2R) signaling to control of intestinal amino acid absorption in mice. METHODS: Absorption, transport, and clearance of essential AAs, specifically lysine, were measured in vivo by Liquid Chromatography triple quadrupole Mass Spectrometry (LC-MS/MS) and ex vivo with Ussing chambers using intestinal preparations from Glp2r+/+ and Glp2r-/- mice. Immunoblotting determined jejunal levels of protein components of signaling pathways (PI3K-AKT, and mTORC1-pS6-p4E-BP1) following administration of GLP-2, protein gavage, and rapamycin to fasted Glp2r+/+ and Glp2r-/- mice. Expression of AA transporters from full thickness jejunum and 4F2hc from brush border membrane vesicles (BBMVs) was measured by real-time PCR and immunoblotting, respectively. RESULTS: Acute administration of GLP-2 increased basal AA absorption in vivo and augmented basal lysine transport ex vivo. GLP-2-stimulated lysine transport was attenuated by co-incubation with wortmannin, rapamycin, or tetrodotoxin ex vivo. Phosphorylation of mTORC1 effector proteins S6 and 4E-BP1 was significantly increased in wild-type mice in response to GLP-2 alone, or when co-administered with protein gavage, and abolished following oral gavage of rapamycin. In contrast, activation of GLP-1R signaling did not enhance S6 phosphorylation. Disruption of GLP-2 action in Glp2r-/- mice reduced lysine transport ex vivo and attenuated the phosphorylation of S6 and 4E-BP1 in response to oral protein. Moreover, the expression of cationic AA transporter slc7a9 in response to refeeding, and the abundance of 4F2hc in BBMVs following protein gavage, was significantly attenuated in Glp2r-/- mice. CONCLUSIONS: These findings reveal an important role for GLP-2R signaling in the physiological and pharmacological control of enteral amino acid sensing and assimilation, defining an enteroendocrine cell-enterocyte axis for optimal energy absorption.

Normalization of obesity-associated insulin resistance through immunotherapy.

Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4(+) T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor V(alpha) repertoires, suggesting antigen-specific expansion. CD4(+) T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic interferon-gamma (IFN-gamma)-secreting T helper type 1 (T(H)1) cells, overwhelming static numbers of T(H)2 (CD4(+)GATA-binding protein-3 (GATA-3)(+)) and regulatory forkhead box P3 (Foxp3)(+) T cells. CD4(+) (but not CD8(+)) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through T(H)2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD3-specific antibody or its F(ab')(2) fragment, reduces the predominance of T(H)1 cells over Foxp3(+) cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4(+) T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.

The essential role of insulin-like growth factor-1 in the intestinal tropic effects of glucagon-like peptide-2 in mice.

BACKGROUND & AIMS: Glucagon-like peptide-2 (GLP-2) is an intestinal hormone that acts through unknown pathways to induce intestinal growth. We investigated the role of the insulin-like growth factors (IGF-1 and IGF-2) as mediators of GLP-2-enhanced growth in the murine intestine. METHODS: IGF-1 expression and secretion were determined in GLP-2-responsive primary intestinal cultures treated with GLP-2. Parameters of intestinal growth were assessed in wild-type (CD1, Igf1(+/+) and Igf2+), heterozygous (Igf1(+/-)), and null (Igf1(-/-) and Igf2(-P)) mice treated chronically with saline, GLP-2, IGF-1, or R-Spondin1. RESULTS: GLP-2 increased IGF-1 messenger RNA expression and IGF-1 secretion in intestinal cultures and increased expression of IGF-1 messenger RNA in mouse small intestine in vivo. Igf1(+/+) and Igf2+ mice responded to .1 microg/g(-1) per day(-1) GLP-2 with increased intestinal weights, morphometric parameters, and proliferative indices. In contrast, Igf1(-/-) mice were unresponsive to the same dose of GLP-2, failing to demonstrate changes in intestinal weight, morphometry, or proliferation. However, a significant effect of 1 microg/g(-1) per day(-1) GLP-2 was observed in Igf1(-/-) mice, but only in terms of small intestinal weight when normalized for body weight. Furthermore, Igf2(-P) mice demonstrated a partially impaired response in terms of small intestinal growth. Both Igf1(-/-) and Igf2(-P) mice exhibited normal-enhanced intestinal growth in response to IGF-1 and/or R-Spondin1. CONCLUSIONS: GLP-2 enhances intestinal IGF-1 expression and secretion, and IGF-1 is required for small and large intestinal growth in response to GLP-2. These findings identify IGF-1 as an essential mediator of the intestinotropic actions of GLP-2.