David B. Rhoads

Vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase and other ATPases

Abstract Vanadate is a potent inhibitor of the Ca2+-ATPase activity of sarcoplasmic reticulum in the presence of A-23187. The purified enzyme is sensitive to vanadate even in the absence of the ionophore. Ca2+ and norepinephrine protect the enzyme against inhibition of vanadate. The nonspecificity of vanadate is emphasized by the finding of inhibition of several other ATPases including the Ca2+Mg2+-ATPases of the ascites and human red cell plasma membranes, Mg2+-ATPase of the ascites plasma membrane, and the K+-ATPases of E. coli and hog gastric mucosal cell membranes. The ascites plasma membrane Ca2+-ATPase (an ecto ATPase) and mitochondrial ATPase are not inhibited by vanadate.

Regulation of glucose transporters in LLC-PK1 cells: effects of D-glucose and monosaccharides.

Regulation of D-glucose transport in the porcine kidney epithelial cell line LLC-PK1 was examined. To identify the sodium-coupled glucose transporter (SGLT), we cloned and sequenced several partial cDNAs homologous to SGLT1 from rabbit small intestine (M. A. Hediger, M. J. Coady, T. S. Ikeda, and E. M. Wright, Nature (London) 330:379-381, 1987). The extensive homology of the two sequences leads us to suggest that the high-affinity SGLT expressed by LLC-PK1 cells is SGLT1. SGLT1 mRNA levels were highest when the D-glucose concentration in the culture medium was 5 to 10 mM. Addition of D-mannose or D-fructose, but not D-galactose, in the presence of 5 mM D-glucose suppressed SGLT1 mRNA levels. SGLT1 activity, measured by methyl alpha-D-glucopyranoside uptake, paralleled message levels except in cultures containing D-galactose. Therefore, SGLT1 gene expression may respond either to the cellular energy status or to the concentration of a hexose metabolite(s). By isolating several cDNAs homologous to rat GLUT-1, we identified the facilitated glucose transporter in LLC-PK1 cells as the erythroid/brain type GLUT-1. High-stringency hybridization of a single mRNA transcript to the rat GLUT-1 cDNA probe and failure to observe additional transcripts hybridizing either to GLUT-1 or to GLUT-2 probes at low stringency provide evidence that GLUT-1 is the major facilitated glucose transporter in this cell line. LLC-PK1 GLUT-1 mRNAs were highest at medium D-glucose concentrations of less than or equal to 2 mM. D-Fructose, D-mannose, and to a lesser extent D-galactose all suppressed GLUT-1 mRNA levels. Since the pattern of SGLT1 and GLUT-1 expression differed, particularly in low D-glucose or in the presence of D-galactose, we suggest that the two transporters are regulated independently.

Rapid upregulation of sodium-glucose transporter SGLT1 in response to intestinal sweet taste stimulation.

Objective:We set out to examine the short-term regulation of the intestinal sodium/glucose cotransporter SGLT1 by its substrate glucose and sweet taste analogs. Summary Background Data:Intestinal SGLT1 is a putative target for antidiabetic therapy; however, its physiological regulation is incompletely understood, limiting its application as a pharmacological target. While it is clearly regulated by dietary composition over a period of days, its short-term regulation by nutrients is unknown. Methods:Sprague-Dawley rats were anesthetized, and the duodenum cannulated. d-glucose, d-fructose, saccharin, d-mannitol, and water were infused for 3 hours, before harvest of proximal jejunum for SGLT1 analysis with Western blotting and quantitative polymerase chain reaction. In further experiments, the receptor region was identified by d-glucose infusion of isolated regions. Lastly, the vagus was de-afferented with capsaicin, and 5HT3-receptor activation of vagal afferents inhibited using ondansetron, before repeating experiments using water or d-glucose infusion. Results:Infusion of d-glucose led to 2.9-fold up-regulation in SGLT1 compared with water or iso-osmotic d-mannitol; this effect was replicated by d-fructose or saccharin. This response was strongest following isolated infusions of duodenum and proximal jejunum, with a blunted effect distally; topography matched the expression profile of sweet taste receptor T1R2/T1R3. The reflex was abolished by capsaicin pretreatment, and blunted by ondansetron. Conclusions:The agonist response implicates the luminal-based sweet-taste receptor T1R2/T1R3, with the reflex apparently involving vagal afferents. The proximal nature of the sensor coincides with the excluded biliopancreatic limb in Roux-en-Y gastric bypass, and this may provide a novel explanation for the antidiabetic effect of this procedure.

Discrimination between Rb+ and K+ by Escherichia coli.

1. The K+ requirment of Escherichia coli is only partially fulfilled by Rb+. The molar growth yield on Rb+ was about 5% of that on K+ and the growth rate in Rb+-supplemented media is lower thatn in K+ influx by any of the four K+ transport systems of E. coli. The high-affinity Kdp system (Km = 2 micron) is poorly traced by 86Rb+. It discriminates against a 86Rb+ tracer at least 1000-fold. The two moderate affinity systems, the high-rate TrkA system (Km = 1.5 mM) and the moderate rate TrkD system (Km = 0.5 mM), discriminate against a 86Rb+ tracer by approximately 10-fold and 25-fold, respectively. 86Rb+ is preferred by the low-rate TrkF system and overestimates its K+ influx by 40%.

Diurnal rhythmicity in glucose uptake is mediated by temporal periodicity in the expression of the sodium-glucose cotransporter (SGLT1)

BACKGROUND Intestinal transport exhibits distinct diurnal rhythmicity. Understanding the mechanisms behind this may reveal new therapeutic strategies to modulate intestinal function in disease states such as diabetes and obesity, as well as short bowel syndrome. Although diurnal rhythms have been amply documented for several intestinal transporters, the complexity of transepithelial transport has precluded definitive attribution of rhythmicity in glucose uptake to a single transporter. To address this gap, we assessed temporal changes in glucose transport mediated by the Na(+)/glucose cotransporter SGLT1. METHODS SGLT1 expression was assessed at 4 times during the day: ZT3, ZT9, ZT15, and ZT21 (ZT, Zeitgeber time; lights on at ZT0; n = 8/ time). SGLT1 activity, which is defined as glucose uptake sensitive to the specific SGLT1 inhibitor phloridzin, was measured in everted intestinal sleeves. Changes in Sglt1 expression were assessed by real-time polymerase chain reaction (PCR) and immunoblotting. RESULTS Glucose uptake was significantly higher at ZT15 in jejunum (P < 0.05 vs ZT3). Phloridzin significantly reduced glucose uptake and completely abolished its rhythmicity. Sglt1 mRNA levels were significantly greater at ZT9 and ZT15 in jejunum and ileum, respectively (P < 0.05 vs ZT3), whereas SGLT1 protein levels were significantly greater at ZT15 in jejunum (P < 0.05 vs ZT3). CONCLUSIONS Our results definitively link diurnal changes in intestinal glucose uptake capacity to changes in both SGLT1 mRNA and protein. These findings suggest that modulation of transporter expression would enhance intestinal function and provide an impetus to elucidate the mechanisms that underlie diurnal rhythmicity in transcription. Modulation of intestinal function would benefit the management of malnutrition as well as diabetes and obesity.

Insulin cessation and diabetes remission after bariatric surgery in adults with insulin-treated type 2 diabetes.

OBJECTIVE The impact of bariatric surgeries on insulin-treated type 2 diabetes (I-T2D) in the general population is largely undocumented. We assessed changes in insulin treatment after bariatric surgery in a large cohort of I-T2D patients, comparing Roux-en-Y gastric bypass surgery (RYGB) with laparoscopic adjustable gastric banding (LAGB), controlling for differences in weight loss between procedures. RESEARCH DESIGN AND METHODS Of 113,638 adult surgical patients in the Bariatric Outcomes Longitudinal Database (BOLD), 10% had I-T2D. Analysis was restricted to 5,225 patients with I-T2D and at least 1 year of postoperative follow-up. Regression models were used to identify factors that predict cessation of insulin therapy. To control for differences in weight loss patterns between RYGB and LAGB, a case-matched analysis was also performed. RESULTS Of I-T2D patients who underwent RYGB (n = 3,318), 62% were off insulin at 12 months compared with 34% (n = 1,907) after LAGB (P < 0.001). Regression analysis indicated that RYGB strongly predicted insulin cessation at both 1 and 12 months postoperatively. In the case-matched analysis at 3 months, the proportion of insulin cessation was significantly higher in the RYGB group than in the LAGB group (P = 0.03), and the diabetes remission rate was higher at all time points after this surgery. RYGB was a weight-independent predictor of insulin therapy cessation early after surgery, whereas insulin cessation after LAGB was linked to weight loss. CONCLUSIONS I-T2D patients have a greater probability of stopping insulin after RYGB than after LAGB (62% vs. 34%, respectively, at 1 year), with weight-independent effects in the early months after surgery. These findings support RYGB as the procedure of choice for reversing I-T2D.

MicroRNA mir-16 is anti-proliferative in enterocytes and exhibits diurnal rhythmicity in intestinal crypts

BACKGROUND AND AIMS The intestine exhibits profound diurnal rhythms in function and morphology, in part due to changes in enterocyte proliferation. The regulatory mechanisms behind these rhythms remain largely unknown. We hypothesized that microRNAs are involved in mediating these rhythms, and studied the role of microRNAs specifically in modulating intestinal proliferation. METHODS Diurnal rhythmicity of microRNAs in rat jejunum was analyzed by microarrays and validated by qPCR. Temporal expression of diurnally rhythmic mir-16 was further quantified in intestinal crypts, villi, and smooth muscle using laser capture microdissection and qPCR. Morphological changes in rat jejunum were assessed by histology and proliferation by immunostaining for bromodeoxyuridine. In IEC-6 cells stably overexpressing mir-16, proliferation was assessed by cell counting and MTS assay, cell cycle progression and apoptosis by flow cytometry, and cell cycle gene expression by qPCR and immunoblotting. RESULTS mir-16 peaked 6 hours after light onset (HALO 6) with diurnal changes restricted to crypts. Crypt depth and villus height peaked at HALO 13-14 in antiphase to mir-16. Overexpression of mir-16 in IEC-6 cells suppressed specific G1/S regulators (cyclins D1-3, cyclin E1 and cyclin-dependent kinase 6) and produced G1 arrest. Protein expression of these genes exhibited diurnal rhythmicity in rat jejunum, peaking between HALO 11 and 17 in antiphase to mir-16. CONCLUSIONS This is the first report of circadian rhythmicity of specific microRNAs in rat jejunum. Our data provide a link between anti-proliferative mir-16 and the intestinal proliferation rhythm and point to mir-16 as an important regulator of proliferation in jejunal crypts. This function may be essential to match proliferation and absorptive capacity with nutrient availability.

Acid hydrolases in neuronal and glial enriched fractions of rat brain

Fractions enriched in neuronal and glial cells from rat brain were assayed for several lysosomal acid hydrolases, namely, β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23), β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21), α-galactosidase (α-D-galactoside galactohydrolase, EC 3.2.1.22), α-mannosidase (α-D-mannoside mannohydrolase, EC 3.2.1.24), N-acetyl-β-glucosaminidase (β-2-acetamido-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30), N-acetyl-β-galactosaminidase, acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2), β-glucuronidase (β-D-glucuronide glucuronohydrolase, EC 3.2.1.31), arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1), glucocerebrosidase and galactocerebrosidase. The data do not show any significant difference in the distribution of these activities between the two cell types.

Restricted Feeding Phase Shifts Clock Gene and Sodium Glucose Cotransporter 1 (SGLT1) Expression in Rats

The intestine exhibits striking diurnal rhythmicity in glucose uptake, mediated by the sodium glucose cotransporter (SGLT1); however, regulatory pathways for these rhythms remain incompletely characterized. We hypothesized that SGLT1 rhythmicity is linked to the circadian clock. To investigate this, we examined rhythmicity of Sglt1 and individual clock genes in rats that consumed food ad libitum (AL). We further compared phase shifts of Sglt1 and clock genes in a second group of rats following restricted feeding to either the dark (DF) or light (LF) phase. Rats fed during the DF were pair-fed to rats fed during the LF. Jejunal mucosa was harvested across the diurnal period to generate expression profiles of Sglt1 and clock genes Clock, Bmal1 (brain-muscle Arnt-like 1), ReverbA/B, Per(Period) 1/2, and Cry (Cryptochrome) 1/2. All clock genes were rhythmic in AL rats (P < 0.05). Sglt1 also exhibited diurnal rhythmicity, with peak expression preceding nutrient arrival (P < 0.05). Light-restricted feeding shifted the expression rhythms of Sglt1 and most clock genes (Bmal1, ReverbA and B, Per1, Per2, and Cry1) compared with dark-restricted feeding (P < 0.05). The Sglt1 rhythm shifted in parallel with rhythms of Per1 and ReverbB. These effects of restricted feeding highlight luminal nutrients as a key Zeitgeber in the intestine, capable of simultaneously shifting the phases of transporter and clock gene expression, and suggest a role for clock genes in regulating Sglt1 and therefore glucose uptake. Understanding the regulatory cues governing rhythms in intestinal function may allow new therapeutic options for conditions of dysregulated absorption such as diabetes and obesity.

Diurnal expression of the rat intestinal sodium-glucose cotransporter 1 (SGLT1) is independent of local luminal factors.

BACKGROUND The intestinal sodium-glucose cotransporter 1 (SGLT1) is responsible for all secondary active transport of dietary glucose, and it presents a potential therapeutic target for obesity and diabetes. SGLT1 expression varies with a profound diurnal rhythm, matching expression to nutrient intake. The mechanisms entraining this rhythm remain unknown. We investigated the role of local nutrient signals in diurnal SGLT1 entrainment. METHODS Male Sprague-Dawley rats, which were acclimatized to a 12:12 light:dark cycle, underwent laparotomy with formation of isolated proximal jejunal loops (Thiry-Vella loops). Animals were recovered for 10 days before harvesting at 4 6-h intervals (Zeitgeber times ZT3, ZT9, ZT15, and ZT21, where ZT0 is lights on; n = 6-8). SGLT1 expression was assessed in protein, and mRNA extracts of mucosa were harvested from both isolated loops (LOOP) and remnant jejunum (JEJ). RESULTS Isolated loops were healthy but atrophic with minimal changes to villus architecture. A normal anticipatory rhythm was observed in Sglt1 transcription in both LOOP and JEJ, with the peak signal at ZT9 (2.7-fold, P < .001). Normal diurnal rhythms were also observed in the protein signal, with peak expression in both LOOP and JEJ at ZT9 to 15 (2.1-fold, P < .05). However, an additional more mobile polypeptide band was also observed in all LOOP samples but not in JEJ samples (61 kDa vs 69 kDa). Enzymatic deglycosylation suggested this to be deglycosylated SGLT1. CONCLUSION The persistence of SGLT1 rhythmicity in isolated loops indicates that diurnal induction is independent of local luminal nutrient delivery, and it suggests a reliance on systemic entrainment pathways. However, local luminal signals may regulate glycosylation and, therefore, the posttranslational handling of SGLT1.