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Graphical abstract V1

Reprogrammed Stomach Tissue As A Renewable Source Of Functional β Cells For Blood Glucose Regulation

Original article published in Cell Stem Cell on February 18, 2016. Click here to read the original article.

Graphical Abstract:
Graphical abstract V1Authors: Chaiyaboot Ariyachet, Alessio Tovaglieri, Guanjue Xiang, Jiaqi Lu, Manasvi S. Shah, Camilla A. Richmond, Catia Verbeke, Douglas A. Melton, Ben Z. Stanger, David Mooney, Ramesh A. Shivdasani, Shaun Mahony, Qing Xia, David T. Breault, Qiao Zhou

Correspondance: qiao_zhou@harvard.edu

In Brief: Ariyachet et al. show that the antral stomach region of the gastrointestinal tract is particularly amenable to being reprogrammed to a β cell fate because of transcriptional similarity and that bioengineered stomach mini-organs containing reprogrammed cells can rescue hyperglycemia when transplanted into diabetic mice.



  • Antral stomach cells reprogram effectively to insulin+
    pancreatic β-like cells
  • Antral endocrine cells are transcriptionally related to
    pancreatic β cells
  • Induced insulin+ cells reverse hypoglycemia after
    transplantation in diabetic mice
  • Reprogrammed cells in bioengineered mini-organs give
    functional rescue in vivo


The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin+ cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin+ cells with close molecular and functional similarity to β cells. Induced GI insulin+ cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control.



Major progress has been made in recent years to produce functional insulin+ cells for cell replacement therapies to treat diabetes. These regenerative technologies include directed differentiation of embryonic stem cells and direct conversion from non-β cells such as liver cells, acinar cells, and others (Hebrok, 2012, Johannesson et al., 2015, Nostro and Keller, 2012, Schiesser and Wells, 2014, Zhou and Melton, 2008). However, because ongoing pathological conditions in diabetes inflict continued damage to native and transplanted β cells (Azzi et al., 2010, Butler et al., 2003, Lakey et al., 2006, Rahier et al., 2008), it is desirable to develop a regenerative system where β cells can be produced in a renewable fashion to counteract β cell loss. The gastrointestinal (GI) tissues are potential sources for such continued generation of β cells. The stomach and intestine are unique among endodermal organs in that they harbor large numbers of adult stem/progenitor cells that constantly produce epithelial cells, including hormone-secreting enteroendocrine cells (Barker et al., 2007, Barker et al., 2010, May and Kaestner, 2010, Schonhoff et al., 2004a). Both organs are developmentally related to the pancreas, arising in adjacent embryonic domains (Offield et al., 1996). Development of gut enteroendocrine and pancreatic endocrine cells also depends on common critical factors, such as Ngn3 (also known as Neurog3) (Gu et al., 2002, Jenny et al., 2002, Lee et al., 2002). Recent studies showed that intestinal cells could be converted into insulin+ cells with either endocrine-specific deletion of FoxO1 or ubiquitous expression of NPM reprogramming factors (Ngn3, Pdx1, and Mafa) (Bouchi et al., 2014, Chen et al., 2014, Talchai et al., 2012a). Although these studies revealed the feasibility of deriving β-like cells from the intestine, critical barriers remain in developing these approaches into future regenerative therapies. FoxO1 plays a critical role in protecting β cells from cellular stress (Kitamura et al., 2005, Talchai et al., 2012b), and deletion or suppression of FoxO1 in pancreatic β cells could result in β cell failure (Talchai et al., 2012b, Talchai and Accili, 2015). Moreover, although NPM factors induce insulin+ cells in the intestine, the induced cells appear to lack certain important β cell genes such as Nkx6.1 and exhibit reduced glucose responsiveness compared with pancreatic β cells (Chen et al., 2014).

We sought to devise improved strategies to derive functional insulin-secreting (insulin+) cells from GI tissues and to harness the regenerative capacity of these tissues as a renewable source of β cells. We report the surprising finding that NPM factors reprogram enteroendocrine cells from the antral stomach more efficiently into functional insulin+ cells compared with enteroendocrine cells from the intestine. Induced antral insulin+ cells also express key β cell factors, including Nkx6.1 and Prohormone convertase 2 (PC2), which intestinal insulin+ cells lack. Our data reveal that native antral enteroendocrine cells share a surprising level of transcriptional similarity with pancreatic β cells. Further, the intestine-specific Cdx2 gene can block efficient β cell reprogramming. Thus, intrinsic molecular differences between antral stomach and intestinal enteroendocrine cells could contribute to the differential reprogramming outcomes. To explore the therapeutic potential of gastric tissue as a source of inducible β cells, we created bioengineered stomach mini-organs; upon transplantation and sphere formation, these structures produced renewable insulin+ cells that reverse hyperglycemia in vivo. Our studies reveal antral stomach tissue as a previously unrecognized source that is highly amenable to reprogramming toward functional insulin+ cells. We also provide proof of principle evidence that bioengineered gastric tissue could serve as a renewable source of β cells for glycemic control.


NPM Factors Efficiently Reprogram GI Enteroendocrine Cells to Insulin+ Cells, with Antral Stomach Showing the Highest Induction Efficiency

Previous studies of reprogramming GI tissues to insulin+ cells have used either deletion of FoxO1 or expression of NPM factors (Ngn3, Pdx1, and Mafa). Surprisingly, no insulin+ cells were reported from stomach with either approach (Chen et al., 2014, Talchai et al., 2012a). To revisit this important question, we performed additional reprogramming experiments in the GI tract. Using adenoviral infection of cultured mouse antral stomach organoids, we observed that the NPM factors are highly effective at inducing insulin expression whereas the other reprogramming factors tested, including Pax4, Insm1, Nkx6.1, and Mafa, are not effective (Figure S1). Based on this observation, we constructed new transgenic mouse lines (TetO-NPMcherry) in which the inducible TetO promoter drives polycistronic expression of NPM factors and the red fluorescent protein Cherry (Figure 1A). Global expression of NPM factors leads to rapid animal death due to hypoglycemia (unpublished observations). To enable long-term observation and comparison of induced insulin+ cells from different GI regions, we targeted NPM factors to the GI enteroendocrine lineage, which shares molecular and developmental similarity with pancreatic endocrine cells (Habib et al., 2012, May and Kaestner, 2010, Schonhoff et al., 2004a), making it an excellent target for β cell conversion. We crossed the TetO-NPMcherry line with the bacterial artificial chromosome (BAC)-transgenic Ngn3-Cre line (Schonhoff et al., 2004b) and the knockin Rosa-floxed-rtTA line (Jackson Laboratory) to derive a triple-transgenic line we call NRT (Figure 1A). The well-described Ngn3-Cre line labels all enteroendocrine cells in the intestine and the majority of antral stomach enteroendocrine cells (Schonhoff et al., 2004b) (Figure S1).

Screen Shot 2016-02-19 at 12.30.54 PM

Figure 1

NPM Factors Efficiently Reprogram Gastrointestinal Endocrine Cells to Insulin+ Cells with the Highest Induction Efficiency in Antral Stomach

(A) Diagram of the triple-cross transgenic mouse line, referred to as NRT (Ngn3-Cre; Rosa-floxed-rtTA; Teto-NMPcherry). Ngn3-cre is used to target inducible expression of the NPM factors (Ngn3, Pdx1, and Mafa) into the enteroendocrine cells of the antral stomach and the intestine. Black bars in Teto-NPMcherry indicate 2A peptides used to mediate polycistronic expression.

(B–G) Doxycycline treatment of NRT animals yielded many insulin+cherry+ cells from the antral stomach (B), the duodenum (C), and the colon (D), among other GI regions. Quantitation showed a higher induction efficiency of insulin+ cells in antrum compared with duodenum (du), ileum (IL), and colon (Co) (E, n = 3 animals, p = 0.0026). Antrum tissue also has higher insulin content (F, n = 3 animals, p = 0.0046). Using FACS-purified cherry+ cells, the expression level of transgenes in the endocrine population was found to be comparable (G), n = 3 animals). Scale bar, 100 μm. Yellow arrows indicate insulin+cherry+ cells; white arrowheads indicate insulincherry+ cells.

See also Figure S1.

After doxycycline (Dox) treatment of NRT animals for 10 days, we observed numerous insulin+ cells in the antral stomach and along the entire length of the intestine (Figures 1B–1D, yellow arrows). The fundus region of the stomach contains relatively few Ngn3+ endocrine cells, and very few of these expressed insulin, suggesting that fundal cells resist NPM-mediated conversion (Figure S1). Quantitative analysis showed significantly higher reprogramming efficiency in the antrum (41.5% ± 8.5%, mean ± SD) than in the proximal (duodenum, 21.4% ± 6.7%), middle (ileum, 14.6% ± 3.3%), or distal (colon, 15.5% ± 3.4%) intestine (Figure 1E). The antral stomach also contains substantially higher levels of insulin protein compared with the intestine (Figure 1F), even though levels of reprogramming factor expression in fluorescence-activated cell sorting (FACS)-purified cherry+ cells from the antrum and different intestinal regions are comparable (Figure 1G).

Enteroendocrine cells in the stomach and intestine include multiple subtypes based on hormone expression (Habib et al., 2012, May and Kaestner, 2010, Schonhoff et al., 2004a). To evaluate whether insulin+ cells are preferentially induced in certain subtypes, we quantified seven major enteroendocrine subtypes before and after induction of insulin+ cells (Figure S1). All endocrine subtypes we examined were reduced upon doxycycline treatment, with the exception of serotonin+ cells, which do not originate from the Ngn3+ lineage (Schonhoff et al., 2004b) (Figure S1). These data indicate that insulin+ cells in both stomach and duodenum arise from multiple endocrine subtypes and/or their common progenitors. We also found the vast majority of induced GI insulin+ cells to be mono-hormonal (Figure S1). These data collectively show that NPM factors can robustly reprogram GI endocrine cells into insulin+ cells, with the highest reprogramming efficiency in the antral stomach.

Induced GI Insulin+ Cells Can Reverse Hyperglycemia Long-Term and Regenerate Rapidly upon Ablation

To test whether the induced GI insulin+ cells can secrete insulin and reverse hyperglycemia, we ablated pancreatic β cells in NRT mice with streptozotocin (STZ), which renders the animals hyperglycemic. Upon Dox treatment and induction of insulin+ cells in the GI tract, hyperglycemia was rapidly reversed and blood glucose levels remained normal for as long as we tracked them (Figure 2A, up to 6 months). In contrast to control animals, which died with hyperglycemia within 8 weeks, nearly every Dox-treated animal was rescued (Figure 2B). Consistent with this effect, intraperitoneal glucose tolerance test (IPGTT) showed substantial improvement after doxycycline induction (Figure 2C) and near-normal blood insulin level in STZ-ablated and Dox-induced animals (Figure 2D).

Fig2 QZ

Figure 2

Induced Insulin+ Cells from the GI Tract Can Reverse Hyperglycemia Long-Term and Regenerate Rapidly

(A) Glucose monitoring of hyperglycemic NRT animals over 6 months. Streptozotocin (STZ) was used to ablate endogenous pancreatic β cells and create hyperglycemia. Doxycycline (Dox) was administered continuously from week 1 onward (red line). Compared with persistent hyperglycemia and death of control animals (−Dox group, black squares), Dox treatment led to long-term suppression of hyperglycemia (+Dox group, red circles). A second round of STZ ablation was conducted at week 10 to evaluate the regenerative capacity of this experimental system. The ensuing hyperglycemia was suppressed again by week 13. Pancreatectomy was performed on week 19 to remove ∼80% of the pancreas. No significant effect on blood glucose levels was observed.

(B–D) Dox treatment and induction of insulin+ cells led to significant improvement in the survival of hyperglycemic NRT animals (B, n = 12 animals in each group). Glucose tolerance tests showed near-normal responses for Dox-treated animals (C, n = 4 animals in each group). The blood insulin levels of the induced animals are comparable with that of wild-type animals and significantly higher than non-induced animals (D, n = 4 animals in each group, p < 0.001).

(E) Immunohistochemistry showed before and after induction of insulin+ cells (first and second panel, respectively). STZ treatment was used at week 11 to ablate the induced insulin+ cells from the GI tract (third panel). Insulin+ cells were regenerated rapidly 3 weeks later (last panel). Ki67 staining labels the proliferating stem/progenitor cell compartment at the base of the glands. Scale bar, 100 μm.

All quantitative data presented as mean ± SD. Statistical significance was evaluated with the Student’s t test (∗∗∗p < 0.001). See alsoFigures S2 and S3.

To confirm that rescue from hyperglycemia results from induction of insulin+ cells in the GI tract, we surveyed insulin expression in other Ngn3-expressing tissues including the brain, testis, and pancreas. No insulin+ cells were found in the brain or testis (data not shown). In the pancreas of NRT animals, STZ treatment led to near complete ablation of endogenous β cells (Figure S2), but Dox treatment induced insulin in glucagon+ cells, which comprise the majority of islet cells after β cell ablation (Figure S2). These glucagon+insulin+ cells do not, however, express other β cell factors such as Glut2 and Nkx6.1, and their insulin expression level is significantly lower than in native β cells (Figure S2). To assess the possibility that these glucagon+insulin+ cells may nevertheless contribute to reversal of hyperglycemia after Dox induction in NRT animals, we resected ∼80% of the pancreas and thus most glucagon+insulin+ cells. No significant changes in blood glucose level followed (Figure 2A). The remnant 20% pancreas showed 0.15 ± 0.03 μg of total insulin (mean ± SD), significantly below the insulin content of antrum (1.89 ± 0.36 μg) or duodenum (1.20 ± 0.63 μg; Figure S4). In comparison, a normal mouse pancreas contains ∼10 μg insulin, although only a fraction of the β cell mass is required to maintain normoglycemia (Bonner-Weir, 2000). These data collectively indicate that induced insulin+ cells from the GI tract are the main source of secreted insulin that led to long-term reversal of hyperglycemia.

The GI tract is a highly regenerative organ, with resident glandular stem cells continuously producing new epithelial cells (Barker et al., 2010, Barker et al., 2007). To evaluate the capacity of GI β cell regeneration from the stem cell compartment, we conducted a second round of STZ treatment (Figures 2A and 2E). Similar to pancreatic β cells, induced insulin+ cells from the antrum and intestine were sensitive to the toxin and disappeared, leading to hyperglycemia (Figures 2A and 2E). However, the diabetic state was again rapidly reversed, concomitant with the reappearance of GI insulin+ cells (Figure 2E). These data illustrate the high regenerative capacity of the genetically engineered GI tissues and their ability to sustain injuries and maintain suppression of hyperglycemia.

We also evaluated the lifespan of antral and intestinal insulin+ cells and their relative contributions toward glycemic control (Figure S3). In a pulse-chase experiment, GI insulin+ cells were first induced by Dox treatment, followed by Dox withdrawal. Intestinal insulin+ cells disappeared within 7 days, whereas stomach insulin+ cells persisted for more than 20 days, consistent with estimated turnover rates of the native intestinal and antral epithelia (Karam and Leblond, 1993, Lehy and Willems, 1976, Messier and Leblond, 1960, Thompson et al., 1990). Antral insulin+ cells continued to suppress hyperglycemia after intestinal insulin+ cells had disappeared (Figure S3). Thus, antral insulin+ cells have a longer lifespan than their intestinal counterparts and can suppress hyperglycemia independently.

Antral Insulin+ Cells Bear Close Molecular and Functional Resemblance to Pancreatic β Cells

Immunohistochemistry revealed that induced insulin+ cells from the antral stomach and the proximal and distal intestine all express β cell factors such as c-peptide, glucose transporter 2 (Glut2, or Slc2a2), prohormone convertase 1/3 (PC1/3), and Pax6 (Figure 3A, quantification shown in Figure S4). However, other key β cell genes, including Nkx6.1, Nkx2.2, and prohormone convertase 2 (PC2), are expressed exclusively or predominantly in antral insulin+ cells (Figure 3A, quantitation shown in Figure S4). qPCR analysis further confirmed that many β cell factors are expressed at substantially higher levels in antral insulin+ cells than in duodenal or colonic insulin+ cells (Figure S4). Endogenous Pdx1, but not endogenous Mafa, is expressed in the native duodenum and antrum (Figure S4), as previously reported (Habib et al., 2012, Offield et al., 1996). Endogenous Mafa is activated strongly in antral insulin+ cells, but only weakly in duodenal and colonic insulin+ cells (Figure S4), whereas endogenous Pdx1 is induced in both antral and intestinal insulin+ cells (Figure S4). In contrast, endogenous Ngn3 is not induced (Figure S4). We observed continued expression of FoxO1 expression in both antral and intestinal insulin+ cells (Figure S4).


Figure 3

Induced Insulin+ Cells from the Antral Stomach More Closely Resemble β Cells Molecularly and Functionally

(A) Immunohistochemistry showed that induced insulin+ cells from the antrum express β cell genes Nkx6.1, Nkx2.2, and Prohormone convertase 2 (PC2), which are largely absent from duodenum and colon insulin+ cells. In contrast, Prohormone convertase 1/3 (PC1/3), glucose transporter 2 (Glut2), and c-peptide (c-ppt) are expressed commonly in antral, duodenal, and colonic insulin+ cells. Arrows indicate antral insulin+ that are PC2+, Nkx2.2+, and Nkx6.1+. Scale bar, 50 μm.

(B) Glucose stimulated insulin secretion (GSIS) in vitro. Antral tissues have significantly higher glucose responsiveness, defined as fold increase of insulin release at high versus low glucose conditions, compared with duodenal and colonic tissues (n = 8, p < 0.001).

(C and D) The antidiabetic drug Glibenclamide (Glib) stimulated insulin release from the antral insulin+ cells whereas Diazoxide (Dzx), a suppressor of insulin release, reduced antral insulin secretion (C, n = 4). In contrast, duodenal and colonic insulin+ cells do not respond to Glib or Dzx (C). Antral insulin+ cells also respond to Exendin-4 (Ex4) with enhanced insulin secretion at high glucose levels whereas duodenal and colonic cells do not respond to Ex4 (D, n = 4).

All quantitative data presented as mean ± SD. Statistical significance was evaluated with the Student’s t test (p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). See also Figure S4.

To assess functional properties of induced insulin+ cells from the stomach and the intestine, we harvested whole epithelial tissues from the antrum, duodenum, and colon of NRT animals after 10 days of Dox treatment. In vitro glucose-stimulated insulin secretion assays were performed with each sample at low-glucose (1.7 mM) and high-glucose (20.2 mM) conditions. Our data showed that although all GI insulin+ cells can respond to high glucose (Figure 3B), the responsiveness of antral insulin+ cells is significantly higher than that of duodenal and colonic insulin+ cells (Figure 3B; data standardized as high-glucose versus low-glucose response ratio: 3.02 ± 0.55 for antrum, 1.65 ± 0.37 for duodenum, and 1.61 ± 0.46 for colon).

To further evaluate the function of induced GI insulin+ cells, we tested their physiological response to glibenclamide (Glib), an anti-diabetic drug that binds to Sur1 and inhibits the ATP-sensitive potassium channel in β cells. Glib treatment led to insulin release from antral, but not from duodenal or colonic, insulin+ cells (Figure 3C). Conversely, treatment with Diazoxide (Dzx), a potassium channel activator, suppressed insulin release from antral insulin+ cells, whereas duodenal and colonic cells showed no response (Figure 3C). Moreover, antral insulin+ cells responded to exendin-4, an antidiabetic drug that activates glucagon-like-peptide receptor (Glp1R), leading to increased insulin release at high glucose concentrations (Figure 3D), whereas duodenal and colonic insulin+ did not respond (Figure 3D). Consistent with these physiological data, antral insulin+ cells express significantly higher levels of Sur1 and Glp1R, compared with duodenal and colonic insulin+ cells (Figure S4).

Thus, molecular and physiological studies together indicate that antral endocrine cells can be reprogrammed efficiently into insulin+ cells that resemble pancreatic β cells, whereas conversion from intestinal endocrine cells is comparatively incomplete.

Native Antral Endocrine Cells Share Substantial Transcriptional Similarity with Pancreatic β Cells

What mechanisms might underlie the significant difference? One long-standing hypothesis postulates that the more transcriptional and epigenetic similarities two cells share, the easier it is to interconvert them (Graf and Enver, 2009, Gurdon and Melton, 2008). Transcriptional studies of specific intestinal endocrine populations have been reported (Egerod et al., 2012, Habib et al., 2012), but transcriptomes of antral endocrine cells remain uncharacterized. We therefore profiled the transcriptomes of enteroendocrine cells from the antrum, duodenum, and colon and assessed their similarity to pancreatic β cells. We used Ngn3-GFP reporter mice to isolate enteroendocrine cells from the different GI regions (Lee et al., 2002); Ngn3 expression in the gut is transient and restricted to endocrine progenitors (Jenny et al., 2002, Lee et al., 2002). Ngn3-GFP labels a mixture of chromogranin− (Chga−) and chromogranin+ (Chga+) cells, representing immature and mature endocrine cells, respectively (Lee et al., 2002) (Figure 4A). Our quantitation showed that the relative proportions of GFP+Chga− and GFP+Chga+ cells are comparable in antral stomach, duodenum, and colon (Figure S5). GFP+ cells purified by FACS from the different GI regions (Figure 4B) constitute ∼1%–2% of the total cell population (Figure 4B), consistent with the estimated prevalence of gut endocrine cells (Schonhoff et al., 2004a).

Fig1 TA

Figure 4

Enteroendocrine Cells of the Antral Stomach Share Substantial Transcriptional Similarity with Pancreatic β Cells

(A) Immunohistochemistry showing distribution of GFP+ in the GI tract of the Ngn3-GFP mouse line. The GFP+ cells include both relatively immature (GFP+Chromogranin) and more mature enteroendocrine cells (GFP+Chromogranin+). Scale bar, 50 μm.

(B and C) Ngn3-GFP+ cells were purified by FACS from antrum, duodenum, and colon (B). Scatterplots of transcriptome comparisons between pancreatic β cells and the GI enteroendocrine populations (C). Antral enteroendocrine cells show a greater similarity with β cells.

(D and E) Analysis of 2,398 β cell-enriched genes showed a general trend of elevated expression in antral enteroendocrine cells compared with duodenal and colonic enteroendocrine cells (D). In particular, antral enteroendocrine cells share a group of genes (group 2) with β cells (D) that are enriched for factors important in β cell development and function (E). Quantitative data presented as mean ± SD. Statistical significance was evaluated with the Student’s t test (p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).

(F) Immunohistochemistry showed that Nkx6.1 is present in a population of Chga+ enteroendocrine cells in the antrum, but not expressed in duodenum or colon (top, arrows). Nkx2.2 is expressed in a majority of Chga+ enteroendocrine cells in the antrum and a minority of Chga+ cells in the duodenum and colon (F, bottom, arrows). Scale bar, 50 μm.

See also Figure S5.

We generated global transcriptome data from the purified cells with Illumina arrays. Comparative analyses showed that endocrine cells from the proximal and distal intestine are more similar to each other and less similar to antral endocrine cells (Figure S5, Spearman correlation coefficients: 0.91 [duodenum versus colon], 0.82 [antrum versus duodenum], and 0.80 [antrum versus colon]). The overall similarity of proximal and distal intestine endocrine cells is high and consistent with published studies (Egerod et al., 2012, Habib et al., 2012) (1,470 differentially expressed genes listed in Table S3). We performed pairwise comparison of the three GI endocrine populations with our published transcriptome data of β cells, which was obtained by FACS purification from the islets of MIP-GFP animals (Li et al., 2014b). This analysis showed overall higher transcriptional similarity between antral and β cells than between intestinal and β cells (Figures 4C and S5, Spearman correlation coefficients: 0.72 [antrum versus β], 0.57 [duodenum versus β], and 0.57 [colon versus β]; Steiger’s Z-test for dependent correlations: p = 6.5 × 10−185). Thus, although enteroendocrine cells from the antrum, duodenum, and colon are more similar to each other than they are to pancreatic β cells, β cells appear to share more transcriptional similarity with antral enteroendocrine cells than intestinal enteroendocrine cells.

To evaluate the expression of β cell-enriched genes in GI enteroendocrine cells, we focused analysis on a collection of 2,398 genes that show higher expression in β cells than in acinar cells (Li et al., 2014b). Antral enteroendocrine cells showed higher expression of many β cell-enriched genes (Figure 4D, group 2 and 3 genes; Table S2) compared with intestinal enteroendocrine cells. In particular, many genes critical for β cell development and function, such as Nkx6.1, Nkx2.2, NeuroD1, Isl1, Rfx6, Insm1, Sur1 (ABCC8), and Glucokinase (GCK), are enriched in antral, compared with duodenal or colonic, enteroendocrine cells (Figures 4E and S5). Immunohistochemistry showed Nkx6.1 expression in a subset of antral GFP+ cells (24.9% ± 3.5%, mean ± SD), but not in the duodenum or colon (Figure 4F). The vast majority of antral Nkx6.1+ cells also express Chga (94.7% ± 3.1%) (Figures 4F and S5). Nkx2.2 is expressed in a majority of GFP+ cells in the antrum (57.2% ± 4.7%), but only in a minority of duodenal (18.7% ± 2.7%) or colonic (23.4% ± 4.3%) Chga+ cells (Figure 4F). Most Nkx2.2+ cells express Chga (69.4% ± 3.4%, 64.0% ± 5.1%, and 65.5% ± 8.3% in antrum, duodenum, and colon, respectively) (Figure 4F; Figure S5). Gene Ontology analyses show that whereas enteroendocrine cells from all GI regions are enriched for pathways involved in regulation of hormone secretion, G-protein-coupled receptor signaling, and vesicle-mediated transport, antral enteroendocrine cells are enriched specifically for the “glucose homeostasis” module (Figure S5; Table S2). Together, these studies reveal a surprising intrinsic difference between endocrine cell populations from the antral stomach and intestine, which likely contributes to their differential capacity for β cell reprogramming.

The Intestine-Specific Gene Cdx2 Can Inhibit β Cell Conversion

In a prior study of acinar to β cell conversion, we showed that persistent expression of acinar cell fate regulators Ptf1a and Nr5a2 blocks acquisition of β cell fate (Li et al., 2014c). Cdx2 is an intestine-specific master regulator gene (Gao et al., 2009), and its persistent expression in intestinal insulin+ cells (Figure 5A) raises the question of whether Cdx2 might block intestinal cells from adopting more complete β cell features. To test this hypothesis, we generated epithelial organoids from the antrum and duodenum of double transgenic Rosa-rtTA;TetO-NPMcherry (Rosa-NPM) animals and treated them with Dox in culture. Similar to our observations in vivo, antral organoids produced more C-peptide+ cells with higher levels of β cell factors compared with intestinal organoids (Figure S6). Next, we expressed either the control cherry gene or Cdx2 using adenoviral infection in the double-transgenic antral organoids (Figures 5B and 5C), followed by treatment with Dox to activate β cell conversion. Cdx2 significantly suppressed expression of multiple β cell genes, including NeuroD1, Nkx2.2, and Nkx6.1 (Figure 5D).


Figure 5

The Intestine-Specific Cell Fate Regulator Cdx2 Can Inhibit β Cell Conversion

(A) Duodenal and colonic insulin+ cells express Cdx2, the master regulator of intestine cell fate whereas antral stomach cells do not express Cdx2 before or after induction in NRT animals. Scale bar, 50 μm.

(B–D) Epithelial organoids were established from antral tissues of double-transgenic Rosa-rtTA;Teto-NPMcherry (Rosa-NPM) animals and infected with either control adenovirus expressing Cherry (pAd-cherry) or adenovirus expressing Cdx2 and Cherry (pAd-Cdx2.cherry) (B and C). Dox treatment was subsequently used to induce β cell conversion in these antral organoids. qPCR analysis showed that ectopic Cdx2 suppressed the expression of multiple β cell genes (D, n = 3). Scale bar, 100 μm.

(E–G) Duodenal organoids were established from Cdx2fl/+ and Cdx2fl/fl animals (E, left and right, respectively). Infection with an adenovirus co-expressing both NPM factors and the Cre recombinase led to simultaneous deletion of floxed Cdx2 allele and expression of NPM factors (E). Complete removal of Cdx2 was observed in majority of Cdx2fl/fl duodenal cells by immunohistochemistry and qPCR analysis (E and F) and led to enhanced expression of multiple β cell genes from the duodenal organoids (G, n = 3). Scale bar, 100 μm.

Quantitative data presented as mean ± SD. Statistical significance was evaluated with the Student’s t test (p < 0.05, ∗∗p < 0.01, and∗∗∗p < 0.001). See also Figure S6.

To further evaluate the role of Cdx2 in intestine reprogramming, we deleted Cdx2 from duodenal organoids. We established duodenal organoids from animals where a single allele or both alleles of the Cdx2 gene are floxed (Figure 5E, Cdx2fl/+ and Cdx2fl/fl). Infection with an polycistronic adenovirus expressing NPM factors and the Cre recombinase (pAd-NPM.Cre) led to simultaneous removal of the floxed Cdx2 allele(s) and expression of NPM factors (Figure 5E). Immunohistochemistry and qPCR confirmed complete removal of Cdx2 from the majority of Cdx2fl/fl duodenal cells (Figures 5E and 5F). Cdx2 deletion significantly enhanced expression of several β cells genes, including Insulin1, insulin2, Nkx6.1, and NeuroD. These data together suggest that Cdx2 acts as a molecular barrier to β cell conversion; thus, failure to downregulate Cdx2 in intestinal insulin+ cells likely contributes to their incomplete acquisition of β cell properties.

Constructing Bioengineered Stomach and Intestine Mini-organs to Produce Insulin+ Cells

Among GI tissues, antral stomach is a superior source of functional β cells by NPM-mediated conversion, and antral insulin+ cells are rapidly replenished from the native stem cell compartment. However, inducing β cells from the native GI tract in situ may have limitations in therapy, because the native endocrine populations regulate many physiological processes (Field et al., 2010, May and Kaestner, 2010, Schonhoff et al., 2004a), and diverting them into β cells may disrupt normal endocrine homeostasis. Moreover, induced β cells positioned along the native GI epithelium may inadvertently respond to dietary as well as blood glucose. To circumvent these potential barriers to therapeutic application, we studied the feasibility of constructing “stomach mini-organs” that contain genetically engineered antral tissues as a reservoir of new β cells.

Following published protocols on bioengineering stomach (Maemura et al., 2004, Speer et al., 2011), we embedded gastric gland units from the antrum of CAGrtTA::TetO-NPMcherry (CAG-NPM) animals in Matrigel, loaded them onto poly(glycolic acid) (PGA) scaffolds, and transplanted the material into the omental flap of immunodeficient NSG recipient animals (Figures 6A–6C). Four weeks later, bioengineered stomach spheres measuring 0.5 to 1 cm in diameter formed outside the native gut (Figure 6D). By histology, 5 out of 15 such spheres showed robust epithelial reconstitution, while the others showed little or no epithelium (Figure S7). Antral glands in the native stomach are composed largely of mucous and endocrine cells and lack acid-secreting parietal cells. The engineered stomachs also showed a simple organization, with one or several layers of Ecadherin+ cells surrounded by connective tissue (Figure S7). The epithelial component contained Sox9+ stem/progenitor cells (Furuyama et al., 2011), Mucin5+ secretory cells, and Chga+ endocrine cells (Figure S7). In parallel, we used a similar bioengineering approach to construct “intestine mini-organs” using duodenal gland units. The success rate for epithelial reconstitution was lower in intestinal spheres (3 out of 15), which contained Muc2+ secretory cells and Chga+ endocrine cells, similar to the native duodenal epithelium. Our observations are consistent with other published studies on bioengineered stomach and intestine (Maemura et al., 2004, Speer et al., 2011).

Fig4. TA

Figure 6

Construction of Bioengineered Stomach and Intestine Mini-organs to Produce Insulin+ Cells

(A–K) Schematic diagram of engineering stomach and intestine mini-organs (A). Gastric or intestinal units were isolated from the antrum or duodenum of CAG-NPM (Cag-rtTA::TetO-NPMcherry) animals (B and I) and loaded onto polyglycolic acid scaffolds (C and J). The scaffolds were placed inside the omental flap of recipient immune-deficient NSG animals. 4 weeks later, an engineered stomach (E. St) or intestine (E. Int) sphere formed (D and K, circled tissue). Scale bars represent 400 μm (B and I) and 6 mm (C and J).

(E–R) In engineered stomach and intestine spheres where reconstitution of epithelium was successful, Dox treatment led to induction of many insulin+ cells (E and L). The induction efficiency is higher for stomach tissues (P, n = 3). Stomach tissues also have higher insulin content (Q, n = 3). The majority of insulin+ cells from engineered stomach express Nkx6.1, PC1/3, and Glut2 (F, G, H, and R) whereas insulin+ cells from engineered intestine lack Nkx6.1 and have reduced PC1/3 expression (M, N, O, and R). Quantitation presented as mean ± SD. Statistical significance was evaluated with the Student’s t test (∗∗p < 0.01 and ∗∗∗p < 0.001).

See also Figure S7.

To evaluate induction of insulin+ cell in the engineered stomach and intestine spheres, we administered Dox for 2 weeks. Many insulin+ cells appeared in the epithelial layer of stomach as well as intestinal spheres. The stomach spheres had significantly more insulin+ cells, higher reprogramming efficiency, and higher insulin content per milligram of tissue (Figures 6E, 6L, 6P, and 6Q). The majority of stomach insulin+ cells express Nkx6.1, Glut2, and PC1/3, whereas intestine insulin+ cells lack Nkx6.1 and have reduced PC1/3 expression (Figures 6F–6H, 6M–6O, and 6R).

Transplanted Stomach Mini-organs Can Control Hyperglycemia in Diabetic Mice

To assess if β cells induced in the engineered stomachs could release functional insulin, we ablated pancreatic β cells in transplanted animals using STZ and then induced insulin+ cells in the engineered stomach spheres by administering Dox (Figure 7A). Of the 22 treated animals, 5 showed sustained decreases in blood glucose levels after Dox treatment (group 1), whereas the others remained hyperglycemic (group 2) (Figure 7B). We monitored animals for 6 weeks and subsequently removed the grafted stomach spheres from G1 mice, which restored hyperglycemia (Figure 7B). Engineered stomach spheres from G1 animals showed good epithelial structures containing many insulin+ cells (Figure S7), whereas spheres from the G2 groups showed limited epithelial structures with few insulin+ cells (Figure S7). Consistent with the glucose monitoring data and histology, G1 animals showed improved responses to intraperitoneal glucose challenge (Figure 7C). Blood insulin levels in G1 animals also were substantially higher than in G2 animals (Figure 7D).

Fig7. new

Figure 7

Transplanted Stomach Mini-organs Can Reverse Hyperglycemia in Diabetic Mice

(A) Diagram of the experimental design. STZ treatment was used to ablate endogenous β cells in NSG animals transplanted with 4-week-old stomach spheres, followed by continuous Dox treatment of induce insulin+ cells. At the end of the experiment, the engineered stomachs were removed surgically.

(B–D) STZ treatment led to rapid hyperglycemia that persists in the absence of treatment (−Dox group, n = 6, black squares) (B). After Dox treatment, a group of five animals showed prolonged suppression of hyperglycemia (G1 animals, n = 5, red squares), whereas another group of animals remained hyperglycemic (G2 animals, n = 17, blue squares) (B). After 6 weeks, the engineered stomach spheres were removed from the G1 animals, which led to their reversal back to hyperglycemia (B). G1 animals showed improved response in glucose tolerance test (C, n = 4) and substantially higher blood insulin levels (D, n = 4) compared with G2 animals or control STZ-treated animals without Dox induction. Wilde-type control animals in (C) (green squares) are non-STZ-treated animals with intact pancreatic β cell mass. Quantitative data presented as mean ± SEM. Statistical significance was evaluated with the Student’s t test (∗∗∗p < 0.001).

(E–G) Sox9+ and Ki67+ cells are present in the engineered stomach after 4-week Dox treatment (F and G), indicating persistence of stem/progenitor cells. PEcam+ blood vessels are closely associated with insulin+ cells inside the engineered stomach sphere (E). Scale bars, 100 μm. Blue channel, DAPI.

See also Figure S7.

Immunohistochemistry revealed PEcam+ blood vessels closely associated with insulin+ cells in engineered stomach spheres (Figure 7E), consistent with previous observations that induced β cells, similar to endogenous β cells, can secrete VEGF and remodel local vasculature (Zhou et al., 2008). Moreover, large numbers of Sox9+ stem/progenitor cells and Ki67+ proliferating epithelial cells are present in the engineered stomach spheres before and after Dox treatment, indicating persistence of a stem/progenitor compartment (Figures 7F and 7G). These studies collectively indicate that induced insulin+ cells from the bioengineered stomach spheres can release insulin into the circulation and regulate blood glucose levels.


The GI tract is a highly regenerative endodermal organ. We sought to harness this regenerative capacity to create a renewable source of functional insulin+ cells by NPM-mediated reprogramming. Our data show that antral stomach enteroendocrine cells are converted to insulin+ cells more efficiently than intestinal enteroendocrine cells and possess molecular and functional hallmarks of pancreatic β cells. Thus, the antral stomach is a surprisingly good source for reprogrammed insulin+ cells, and we demonstrate the application of bioengineered stomach spheres to control blood glucose levels.

Expression of NPM factors previously led to formation of insulin+ cells in the intestine (Chen et al., 2014). Our experimental system is similar to this previous report and confirms induction of insulin+ cells in the intestine with incomplete β cell conversion. In contrast, antral stomach endocrine cells are more fully reprogrammed, with robust expression of key β cell genes and substantially improved glucose responsiveness. Our studies suggest that the difference can be attributed, at least in part, to intrinsic molecular differences between antral and intestinal enteroendocrine cells. Higher levels of β cell fate regulators in antral enteroendocrine cells may facilitate their conversion, whereas Cdx2, which is specifically expressed in all intestinal, but not stomach, cells inhibits conversion. It is notable that Cdx2 expression persists in induced insulin+ intestinal cells. Prior studies have shown that ectopic Cdx2 expression in stomach promotes an intestine fate (Silberg et al., 2002, Verzi et al., 2013), whereas Cdx2 loss in cultured intestinal organoids activates antral differentiation (Simmini et al., 2014). Continued expression of Cdx2 in intestinal insulin+ cells may thus present a molecular barrier for complete reprogramming.

Compared with the gastric antrum, the gastric corpus contains few Ngn3-derived enteroendocrine cells, and few such cells expressed insulin after NPM induction. Global expression of NPM factors also induced few insulin+ cells in the fundus (Figure S1). Thus, gastric corpus endocrine cells, which are distinct from those in the antrum or intestine (Choi et al., 2014, Li et al., 2014a) and mainly derive from Ngn3-independent lineages (Li et al., 2014a, Schonhoff et al., 2004b), are not amenable to NPM-mediated β cell conversion. What might account for this resistance? The antral stomach shares a close developmental origin with the pancreas, with both organs arising from a common Pdx1+ endodermal domain during embryogenesis (Wells and Melton, 1999). Therefore, we speculate that the epigenetic landscape of endocrine cells from the fundus is more distinct than those from the antrum, making them harder to convert into β cells. Future studies will be necessary to understand these regional distinctions.

FoxO1 deletion also leads to formation of insulin+ cells in the intestine, suggesting a therapeutic path toward inducing insulin+ cells in situ (Bouchi et al., 2014, Talchai et al., 2012a). Our approach offers several advantages. First, with our method, induced insulin+ cells preserve FoxO1 function, which is known to protect β cells from physiologic stress (Kitamura et al., 2005, Talchai et al., 2012b). Second, with bioengineered stomach spheres, native endocrine cell populations in the gut remain undisturbed, and their functions in physiology are preserved. Third, by separating engineered stomachs from the native organ, induced β cells can be positioned to respond only to changes in blood and not luminal glucose levels.

In summary, our study offers a new approach to harness the intrinsic regenerative capacity of the stomach epithelium to replenishing β cell mass in vivo. Given ongoing pathological insults that continuously erode native or transplanted β cells in diabetes, long-term treatment may require repeated transplants. The regenerative system we propose could eliminate that need, and the number and size of transplanted stomach spheres could be manipulated to control β cell numbers. Coupled with recent progress in genome engineering and the ready access to human gastric epithelium from biopsies and differentiated induced pluripotent stem cells or embryonic stem cells (McCracken et al., 2014), the therapeutic applications of this approach are considerable.

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New Class Of Antigens Might Help In Diagnosis And Treatment

New Class Of Antigens Might Help In Diagnosis And Treatment


Original article written by R. Siva Kumar for Counsel & Heal on February 15, 2016. Click here to read the original article.

A novel class of antigens might help scientists to understand why type 1 diabetes develop, finds research from a University of Colorado Denver.

This type of diabetes has the immune system turning against the body’s own tissues. The type 1 diabetes shows that insulin-producing beta cells identified in the pancreas are hit by immune cells, especially T cells.

The insulin is a hormone vital for maintaining blood glucose, and without it, things can become threatening for life. Results can help scientists to invent the first-ever cure for this form of diabetes.

“Our lab studies the type of T cell known as a CD4 T cell,” Kathryn Haskins, corresponding author of the article, said in a press release. “We have focused on autoreactive CD4 T cells using a mouse model of autoimmune diabetes. We have been especially interested in identifying the antigens that activate these T cells.”

Such antigens can also help to find autoreactive T cells in the early stage of the disease and in those who are at risk. It helps in the identification and treatment too. By developing a way of turning off harmful T cells, they may be able to prevent the illness.

There may be some modified peptides that are called “foreign” to the human immune system. Some of these become targets for autoreactive T cells. Other autoimmune diseases can also be understood through them.

The study was published in the Feb. 12, 2016 issue of Science.

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The 2015 Pediatric Research That Should Change Practice

Original article written by Alan Greene, MD and Laurie Scudder, DNP, PNP for Medscape Pediatrics on February 11, 2016. Click here to read the original article.

Editor’s Note:
Keeping up with the relentless body of literature in specialty journals is a daunting task for pediatric primary care providers. Nevertheless, keep up they must; much of this new research affects day-to-day practice and, crucially, early detection and management of numerous conditions that first present in the primary care setting. Medscape spoke with Alan Greene, MD, a Medscape advisor, adjunct professor of pediatrics at Stanford University School of Medicine, and founder and CEO of DrGreene.com, about his picks of the most interesting and important studies in 2015 and their implications for practice.

Medscape: A number of studies in the last 12 months have added yet more urgency to the concerns regarding the dangers of child obesity. A paper[1] relying on data from recent National Health and Nutrition Examination Surveys (NHANES) found that less than 1% of US children had an ideal healthy diet score. Type 2 diabetes rates are rising, and another paper[2] found that long-term complications and mortality were worse in these children than their counterparts with type 1 diabetes. New data show that sweetened beverages, including milk products, are a major contributor to obesity.[3] Yet another study, this one conducted in Iran, found that breastfeeding is protective well into childhood.[4] Despite our growing recognition of factors that contribute to obesity, a solution is still elusive and difficult. What are your key take-away messages from the last years’ worth of research, and how do you suggest making it practical and implementable in the primary care setting?

Dr Greene: The study examining NHANES data[1] is striking in that it was looking for a measurable index of cardiovascular (CV) health in kids so that we could see where we are and where we’re going in the future as lifestyle and interventions change. By and large, the researchers found that the overwhelming majority of babies enter the world with pristine CV systems, with hearts and blood vessels that are supple, powerful, and beautiful. However, by the time they are adolescents, under our watch as pediatricians, the great majority of children have already developed significant CV risk factors.

This is a fairly recent phenomenon. When I started in pediatrics, it was very unusual to see a child with elevated blood pressure. Today, there are millions of children in the United States with elevated blood pressure. It used to be unusual to see kids with elevated serum cholesterol or triglycerides or with a waist size of 36 or 40 inches. It was unusual to see kids with elevated glucose unless they had type 1 diabetes.

Today, two thirds of American middle school and high school students already have at least one of those conditions that used to wait until middle age. These data add urgency to the obesity epidemic we currently see. It’s a metabolic ticking time bomb.

The study examining children with young-onset diabetes[2] was equally eye-popping. Traditionally, many of us breathe a sigh of relief when we hear that diabetes is type 2 as opposed to type 1. We know the severity of type 1 diabetes in kids. But this study found that type 2 diabetes in childhood and adolescence actually has more morbidity and greater mortality than type 1, the opposite of that traditional wisdom. We should be even more concerned with kids when they develop type 2 diabetes.

This finding was shocking to me, and I pay close attention to this literature. It lends, again, more urgency to the situation. One little side note from this study is about sweetened beverages as a contributor not just to obesity but to type 2 diabetes. We are already aware of the link between obesity and sugary drinks, sugary sodas, juices, and juice-like beverages. But this study found a strong association with chocolate milk and sweetened milk and type 2 diabetes.

Given this new urgency regarding the risks of diseases comorbid with obesity, what do we do? There are a few things. This is a dominant medical issue of our time, and it belongs as part of every well child visit, something that we’re measuring and tracking over time and helping families to learn key messages with every visit. In early childhood, especially in the first couple of years of life, feeding early and often the flavors and textures that you want kids to learn to love can have a profound trajectory on what kids learn to like and is the key to building good food habits from the start. We need to encourage family meals from the beginning, tell parents to not give up on vegetables, introduce a lot of variety, and serve something green at every lunch and every dinner.

Once kids have really set their initial tastes, by around age 2 or 3 years, one of the most effective ways to get them to like better food is to have them involved in the process, to have family meals at home where everybody is eating some of the same food. In particular, get kids involved in meal prep, go to the farmer’s market to select the foods, and, for families that have the option, get kids involved in growing real food. Cooking classes are one of the most fun things that parents can do with children to get them involved in preparing food they will enjoy and that their bodies will also enjoy.

Medscape: Those are recommendations that are easily implemented and understood by families and provide good advice for clinicians practicing in more resourced areas. But what about those kids who live in stressed, urban environments? Are there practical suggestions for those families that we are missing?

Dr Greene: Smartphones are very prevalent in less advantaged communities. One of the key nutritional barriers these families face, families that reside in food deserts where healthy food is difficult to access, is finding interesting and tasty foods among what is available and learning which foods are the healthiest.

One free Smartphone app that I like is Fooducate. The app allows you to scan any barcode, gives you a letter grade (A, B, or C) for how healthy that food is, and suggests others in the same category that might be healthier. The app ranks foods on both flavor and nutrition and can help lead consumers in a positive direction.

Medscape: This sounds like the kind of thing children would find fun to do at a store.

Dr Greene: Exactly. It’s a great way to eliminate food battles at a grocery store. Parents can tell children they can pick anything that’s a B or better.

Sodium: The Underreported Food Concern for Children

Medscape: Another report,[5] also based on data from NHANES, concluded that US school-aged children, on average, consume sodium in excess of recommended levels regardless of age, sex, race/ethnicity, income, or weight status. Unlike the attention paid to sweetened beverages, sodium doesn’t get as much press. What is the current state of evidence regarding hazards of excessive sodium intake in children?

Dr Greene: Sodium often doesn’t get enough attention. To provide some context, in the early 1970s, Finland had the highest rate of hypertension and one of the highest levels of CV disease, heart attacks, and stroke of any country on the planet. As a country, they decided to take that on and implemented a number of population-wide public health measures, and they were able to get their average national blood pressure down to normal. One of the big ones was that they decreased the average sodium intake by over 25% to reach recommended levels. It had a profound national impact. Very seldom do you see such a major public health advance.

The Centers for Disease Control and Prevention estimates that if we could reach the recommended sodium intake level of 2300 mg/person/day in the United States—perhaps even a generous recommended level—that we would save between 280,000 and 500,000 lives over the next 10 years, not to mention the improved health, brain health, and physical activity that could be had. It’s a big issue. This study published in the MMWR looked at sodium intake among school-aged kids and found that the average daily sodium consumption in children was 3279 mg/day, almost 1000 mg/day above the recommended level of 2300 mg/day. Consumption is even higher among high school students. Most of the sodium intake comes from 10 food categories together: pizza; breads and rolls; cold cuts; savory snacks; sandwiches; cheese; chicken patties, tenders, or nuggets; pasta mix dishes; Mexican mix dishes, and soups.

Becoming more aware of sources of sodium in the average child’s diet and looking for options that are made flavorful can have a profound impact. Sodium, especially in processed foods, is often hidden. People are aware that there is sodium in their fast food French fries. They may not be aware that the milkshake may have more sodium than the French fries.

Sleep: Kids Are Just Not Getting Enough

Medscape: The importance of sufficient sleep as a major contributor to both physical and psychosocial well-being is increasingly recognized. In late 2014, the American Academy of Pediatrics Adolescent Sleep Working Group issued a policy statement[6] urging schools to consider delaying start time in order to allow middle and high school students to achieve sufficient sleep. Dealing with sleep and sleep-related problems, however, can be daunting, particularly in a primary care setting with limited time. Can you summarize the latest research examining brief interventions that can be deployed in the primary care setting to promote healthy sleep?

Dr Greene: I do think that one of the roles of pediatricians is advocacy. The literature would suggest that delaying school start timesto at least 8:30 in the morning would improve health in a variety of ways, including reductions in obesity, hypertension, and attention-deficit/hyperactivity disorder. It would improve mood, decrease depression and suicidal thoughts or actions, and decrease car accidents in teen drivers.

Where this change has been implemented, that is exactly what has been found. This is an intervention that works, but many school districts are reluctant to adopt this change for a variety of practical reasons. It’s a great issue, and pediatricians should get involved by speaking, writing or calling their local school board, and letting their community know that we as a profession are behind this. The Academy of Pediatrics is behind it, but we as local pediatricians must also say clearly that we are behind it.

As far as things to do in the office, one of my favorites is to try to support the circadian rhythm, so named because it’s “circa dian.” It’s about a day. For most of us, it would be about a 25-hour rhythm where not only do we have sleepiness and arousal that rise and fall, but we also have fluctuations in blood pressure, body temperature, and many hormones. It is a profound rhythm that we share with other living beings that is reset daily by certain cues from the environment. We are seasonal creatures. If we were in a cave and had none of these external cues, our circadian rhythm would eventually get completely off from other people in the external world. But for us, that rhythm is reset by something called zeitgebers.

Zeitgebers are our friends. The more they are in line with each other and the more they are consistent, then the better, longer, and deeper sleep we have. The most profound zeitgeber is probably light. We are very light-sensitive creatures. When we look back before the invention of the electric light bulb, kids tended to sleep like a baby—all night long, soundly, profoundly without waking up, even if there was a loud noise. Today sleeping like a baby often means waking up crying every couple of hours. Sleep for teenagers is often something that’s disrupted as well. Childhood sleep is not as peaceful as it used to be.

One thing that we can do is try to keep the environment as dim as possible between sunset and sunrise. That can have a profound impact on sleep. When you’re camping, you tend to get very drowsy a couple of hours after sunset. That’s difficult in our modern, urban, digital life, but the more we can at least remove the wavelengths of light that trigger melatonin suppression, the easier it is to sleep.

That means paying attention to screens. There are now apps for a variety of different computer and smartphone screens that will pull out the blue wavelength of light, about 475 nm. You can get light bulbs that pull out that wavelength of light in the evening or wear blue-blocker sunglasses to get rid of it. There is a pigment in the retina, melanopsin, that responds to a 475-nm signal and suppresses melatonin or disorganizes it for the rest of the night. Eliminating that sunset to sunrise is a rather simple thing that can help people get drowsy earlier. Part of that means not viewing screens in the last hour or so before bed at least.

Another strong zeitgeber is temperature. For most of the history of humanity, we have experienced our evenings and nights as much cooler than daytime; but with central air and central heating, we have compressed our temperature window in a very narrow range. Creating a cooler nighttime environment, 7 degrees cooler or more, helps with falling and staying asleep.


Iron Status in Infants: Does It Matter?

Medscape: Delaying cord clamping after birth for approximately 3 minutes has been demonstrated to result in improved iron stores in infants up to 4 months of age.[7] A follow-up paper[8] published earlier this year examined the effects of those increased iron stores on neurodevelopment and concluded that the practice benefits fine motor and social skills in early childhood, particularly among boys. This finding is in contrast to a systematic review[9] that concluded that while there may be some evidence demonstrating that routine iron supplementation in children 6-24 months of age may improve hematologic values, evidence of an improvement in clinical outcomes, including developmental outcomes, is lacking. How should pediatric providers be monitoring—and potentially addressing—iron insufficiency in young children?

Dr Greene: The systematic review[9] article earlier this year did suggest that there is not conclusive evidence that testing for iron deficiency anemia by checking hemoglobin and hematocrit and then supplementing with iron does anything to improve developmental outcomes. You can change the hematologic indices, but we may or may not be able to change the cognitive deficits and behavior problems that we do see with iron deficiency.

That review is still somewhat controversial. These researchers were not able to detect a difference in development with screening and subsequent iron supplementation. The US Preventive Services Task Force also now thinks that there is insufficient evidence to recommend routine iron screening.[10] Of note, the issue of iron supplementation was called into question this year with publication of a very interesting paper[7] suggesting a powerful impact from an easier intervention. That is something I would call optimal cord clamping. It’s often called delayed cord clamping in the literature, but I favor the term “optimal.” I don’t think it’s delayed.

The story here is that at the moment a baby is born, about a third of their blood is still circulating in the placenta and umbilical cord. For most of human history, people would watch the cord pulse and pump blood into the baby, largely eliminating iron deficiency anemia. With that blood, the cord would also pump in oxygen, oxygen-carrying capacity in the red blood cells, white blood cells, antibodies, stem cells—a whole host of good things.

But in the early 20th century, the medical community decided to clamp the cord immediately after birth so that we could examine the child. In doing that, we separated ourselves from our history as a species. It’s not just us. There is not one known mammalian species that actively cuts the cord prior to it stopping pulsing.

The idea of waiting an extra maybe 90 seconds to 3 minutes after the baby is born is now supported by published studies looking at the neurodevelopment in kids that had this extra bolus of iron at birth as humans typically had in the past; researchers found improved fine motor and social skills years later, particularly in boys.[11] That’s a zero-cost and easy way to help kids start off with a better iron store.

The American Academy of Pediatrics recommends that exclusively breastfed kids be supplemented with iron.[12] I think that’s a reasonable thing to do, but it kind of raises the question of why? Breast milk is presumably the perfect food, the ideal food for human babies. Why would it not have enough iron? There are two important nutrients that kids seem to need that are not present in breast milk. One of those is vitamin D, and that makes sense because historically we got very little vitamin D from breast milk and a lot of it from the sun. Now that children spend most of their childhood indoors, they need another source.

The second nutrient is iron. Is that because the kids weren’t getting iron supplements or iron-fortified cereals throughout history? I think the reason is that kids historically got a big bolus of iron from a different direction, and restoring that would be a simple way to help improve iron status throughout childhood.

Medscape: You note that the rationale behind earlier cord clamping was to allow examination of the child. Were concerns about maternal-fetal transfusion another reason for implementation of immediate clamping?

Dr Greene: There wasn’t a concern at the time about jaundice or maternal-fetal transfusion. The change happened around 1913. One of the big reasons was a concern about maternal hemorrhage. The thought was that immediate clamping would be safe for the baby and safer for the mom in terms of hemorrhage.

It turns out that earlier clamping did nothing to reduce hemorrhage. And while it did allow quick examination and resuscitation of the child if necessary, you don’t need to clamp the cord to examine and resuscitate a child. The extra blood and oxygen that the infant is getting during that golden minute is exactly what we’d want to be giving them anyway.


What About the Microbiome?

Medscape: The microbiome continues to be a fascinating area of research with better recognition that microbiota are ecologically engineered by mothers and breastmilk. Can you review what we’ve learned in the last year, particularly in regard to the influence of breast milk on both fetal and postnatal development?

Dr Greene: There are a number of risk factors that we know influence the odds that a child will end up developing allergies. Examples are type of delivery (caesarean vs vaginal), exposure to smoking in the household, pets or no pets, birth order of the child, urban vs rural residence, and breast-fed vs formula-fed.

The thing that is striking is that all of those seemingly separate risk factors each have a profound influence on the developing microbiome in the baby. The microbiome may be the common pathway in the development of allergic disease, atopic disease, and eczema.

It is surprising to learn that there are more than 200 ingredients in breast milk that are not digestible and do not directly nourish the baby. Why are they there? Well, it turns out that they cultivate a particular microbiome and nourish the bacteria colonizing the infant’s gut.

There are a growing number of studies that provide evidence of a long shadow from the microbiome that colonizes a child. In particular, some of these studies are looking at the relationship between the type of bacteria with which a child is colonized, short-term symptoms like cough, and long-term symptoms like childhood obesity.

One of the things that we have learned is that, in addition to the gut microbiome that is most familiar to us, there is also a genital microbiome. There is a skin microbiome, varying skin microbiomes, an oral microbiome, and the nasal microbiome.

Recently we’ve learned that the placenta, which we used to think was sterile, has its own microbiome. The developing baby is influenced by a community of bacteria as well. It turns out that the placental microbiome that people thought would be similar to mom’s vaginal microbiome is not, nor is it similar to the gut microbiome. It is actually closest to the oral microbiome for reasons we haven’t really learned yet.

Just prior to birth, the infant gut is sterile. However, the gut is colonized early on, and that early colonization depends on two things. First, it depends on what bacteria the baby is exposed to early on. For a caesarean -section baby, the first bacteria may be mom’s skin bacteria. For a vaginally delivered infant, that exposure may be vaginal and gut bacteria. Maternal stool is often involved in delivery, and that may be a nonaccidental part of the system.

The second factor that affects early infant gut colonization is the food that is nourishing the bacteria. Breast milk contains a certain set of bacteria linked to mom’s gut. Formula contains other, often more inflammatory types of bacteria.

Most of us are aware of the alteration in the gut microbiome associated with antibiotics, but as you know, there are many other factors out there that will disrupt the microbiome. Sometimes those disruptions are inevitable. We’re going to have to alter that gut microbiota, so what are the implications? Is that something that should factor into a decision whether or not to treat a child with an antimicrobial? Where does that leave us with probiotics? Is that something that should be initiated or not? Do we have the answers to those questions yet?

There are a number of studies showing hints of long-term problems with antibiotics. We know that in livestock, antibiotics have been used as growth promoters to help fatten up cattle more quickly. There are some suggestions that antibiotics could also produce obesity in kids.

Disruptions in microbiota may also be associated with autoimmune problems. One recent study this year found an association between antibiotic use and development of arthritis in kids.[13] We just have glimpses of what those connections may be and don’t know the full picture. But we do know that as many as 50%, maybe more, of the antibiotics that are given to children for respiratory conditions are not helpful.[14,15] It makes all the sense in the world to reserve antibiotics for when they are most useful and most necessary.

Clearly antibiotics have been one of the greatest discoveries and inventions in the history of humanity, but reserving them for situations where they’re most necessary only makes them more powerful and can help eliminate some of the unintended consequences we don’t even understand yet.

On the probiotic side, there’s also a lot we don’t know. There have been some studies going back more than 100 years showing that yogurt, for instance, led to improved health and longevity. There have been a number of placebo-controlled, double-blinded studies recently showing specific outcomes from specific strains in probacteria, improvement in things like the length of diarrhea, illness, symptoms of abdominal discomfort, eczema, and likelihood of getting respiratory infections including the flu.

But each of those also is a relatively tiny glimpse into the potential benefits of supporting and cultivating a diverse ecosystem within the gut. Probiotics may be helpful. It’s hard to say at this point which ones would be the best or how much. One thing that we can say is that eating a healthy diet is likely to select for bacteria that thrive on the healthy diet and would be a reinforcing virtuous circle. It would help you crave those foods more and help get the most benefit out of those foods.

Is Looking at Glycemic Index Valid?

Medscape: Are there any other recent studies that you would like to discuss?

Dr Greene: I think one very interesting 2015 study, published in November, looked at the glycemic index.[16] The idea behind the glycemic index is to measure the effect of a particular food or type of food on the subsequent blood glucose curve. Foods with a high glycemic index tend to raise blood sugar a lot. Low glycemic index foods have a much more muted effect on blood glucose. Glycemic index provides one way to look at why a food might be healthier or less healthy. You can find tables listing the glycemic index for a wide variety of foods.

In this study, the researchers measured about 50,000 different meals in 800 subjects and found something that in retrospect should have been obvious but was very surprising to me. The authors concluded that, to a large degree, individual foods do not have their own glycemic index. Rather, the glucose response to a particular food depends on the person. For instance, tomato is considered to be a very low glycemic index food, and yet for one subject in the study, tomatoes specifically prompted high blood glucose levels.

Data from a continuous blood glucose monitor worn by another individual demonstrated a glucose level that shot up to 260 mg/dL after a meal that included brown rice, which is reasonable from a glycemic index perspective. Different foods do, in fact, affect different people differently. We’re at the beginning of being able to figure out what that all means. It is probably a combination of an individual’s genetics and microbiome. Different foods really affect us differently, and the same great diet isn’t good for everyone.


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  3. O’Connor L, Imamura F, Lentjes MA, Khaw KT, Wareham NJ, Forouhi NG. Prospective associations and population impact of sweet beverage intake and type 2 diabetes, and effects of substitutions with alternative beverages. Diabetologia. 2015;58:1474-1483. Abstract
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  16. Zeevi D, Korem T, Zmora N, et al. Personalized nutrition by prediction of glycemic responses. Cell. 2015;163:1079-1094.Abstract


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Scientists Identify Factor That May Trigger Type 1 Diabetes

Original article provided by University of Colorado Denver on February 11, 2016. Click here to read the original article.

A team of researchers, led by investigators at the University of Colorado School of Medicine, have identified a new class of antigens that may be a contributing factor to type 1 diabetes, according to an article published in the current issue of the journal Science.

In autoimmune disease, the key question is why the immune system attacks the body’s own tissues. Type 1 diabetes is the autoimmune form of diabetes, in which insulin-producing beta cells in the pancreas are destroyed by immune cells, especially those known as T cells. Insulin is the hormone that regulates levels of glucose in the blood and without insulin, a life-threatening disease results. Currently, there is no cure for type 1 diabetes.

“Our lab studies the type of T cell known as a CD4 T cell,” said Kathryn Haskins, PhD, professor of immunology and microbiology and corresponding author of the article. “We have focused on autoreactive CD4 T cells using a mouse model of autoimmune diabetes. We have been especially interested in identifying the antigens that activate these T cells.”

Antigens for T cells are pieces of proteins, or protein fragments (peptides) that have to be taken up and presented to the T cells by antigen-presenting cells. Normally, a CD4 T cell is supposed to respond to “foreign” antigens, like a viral peptide. But in autoimmune disease the T cells respond to antigens that are generated in the body. Such proteins and peptides are called autoantigens.

When an autoreactive T cell sees its antigen, it becomes activated and can initiate disease. By identifying those antigens, scientists may be able to use that information to detect autoreactive T cells early in disease, or better yet, in at-risk individuals. If they are able to use the antigens to turn off destructive T cells, they may be able to prevent the disease.

Haskins and others, including fellow corresponding author Thomas Delong, PhD, assistant professor of immunology and microbiology, conducted experiments to analyze the fractions of beta cells that contain antigen for autoreactive CD4 T cells in order to identify autoantigens in type 1 diabetes. They discovered a new class of antigens that consist of insulin fragments fused to peptides of other proteins present in beta cells. That fusion leads to generation of hybrid insulin peptides that are not encoded in an individual’s genome.

If peptides in the body are modified from their original form, they essentially become “foreign” to the immune system and this may explain why they become targets for the autoreactive T cells. The discovery of hybrid peptides as targets of the immune system provides a plausible explanation of how the immune system is tricked into destroying the body’s own beta cells. The discovery may also lead to a better understanding of other autoimmune diseases.

More Information: “Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion,” by T. Delong et al. science.sciencemag.org/cgi/doi/10.1126/science.aad2791

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Role of the integrated stress response in type 1 diabetes pathogenesis
In individuals with type 1 diabetes (T1D), the insulin-producing beta cells are spontaneously destroyed by their own immune system. The trigger that provokes the immune system to destroy the beta cells is unknown. However, accumulating evidence suggest that signals are perhaps first sent out by the stressed beta cells that eventually attracts the immune cells. Stressed cells adapt different stress mitigation systems as an adaptive response. However, when these adaptive responses go awry, it results in cell death. One of the stress response mechanisms, namely the integrated stress response (ISR) is activated under a variety of stressful stimuli to promote cell survival. However, when ISR is chronically activated, it can be damaging to the cells and can lead to cell death. The role of the ISR in the context of T1D is unknown. Therefore, in this DRC funded study, we propose to study the ISR in the beta cells to determine its role in propagating T1D.
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