BMAL1 Regulates Glucokinase Expression Through E-Box Elements In Vitro.

The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.

Portal milieu and the interplay of multiple antidiabetic effects after gastric bypass surgery.

Diabetes is a worldwide health problem. Roux-en-Y gastric bypass (RYGB) leads to rapid resolution of type 2 diabetes (T2D). Decreased hepatic insulin resistance is key, but underlying mechanisms are poorly understood. We hypothesized that changes in intestinal function and subsequent changes in portal venous milieu drive some of these postoperative benefits. We therefore aimed to evaluate postoperative changes in portal milieu. Two rat strains, healthy [Sprague-Dawley (SD)] and obese diabetic [Zucker diabetic fatty (ZDF)] rats, underwent RYGB or control surgery. After 4 wk, portal and systemic blood was sampled before and during an intestinal glucose bolus to investigate changes in intestinal glucose absorption (Gabsorp) and utilization (Gutil), and intestinal secretion of incretins and glucagon-like peptide-2 (GLP-2). Hepatic activity of dipeptidyl peptidase-4 (DPP4), which degrades incretins, was also measured. RYGB decreased Gabsorp in both rat strains. Gutil increased in SD rats and decreased in ZDF rats. In both strains, there was increased expression of intestinal hexokinase and gluconeogenesis enzymes. Systemic incretin and GLP-2 levels also increased after RYGB. This occurred without an increase in secretion. Hepatic DPP4 activity and expression were unchanged. RYGB perturbs multiple intestinal pathways, leading to decreased intestinal glucose absorption and increased incretin levels in both healthy and diabetic animals. In diabetic rats, intestinal glucose balance shifts toward glucose release. The portal vein as the gut-liver axis may integrate these intestinal changes to contribute to rapid changes in hepatic glucose and hormone handling. This fresh insight into the surgical physiology of RYGB raises the hope of less invasive alternatives. NEW & NOTEWORTHY Portal milieu after gastric bypass surgery is an underinvestigated area. Roux-en-Y gastric bypass perturbs multiple intestinal pathways, reducing intestinal glucose absorption and increasing incretin levels. In diabetic rats, the intestine becomes a net releaser of glucose, increasing portal glucose levels. The portal vein as the gut-liver axis may integrate these intestinal changes to contribute to changes in hepatic glucose handling. This fresh insight raises the hope of less invasive alternatives.

Intestinal sweet-sensing pathways and metabolic changes after Roux-en-Y gastric bypass surgery.

Studies suggest that improvements in type 2 diabetes (T2D) post- Roux-en-Y gastric bypass (RYGB) surgery are attributable to decreased intestinal glucose absorption capacity mediated by exclusion of sweet taste-sensing pathways in isolated proximal bowel. We probed these pathways in rat models that had undergone RYGB with catheter placement in the biliopancreatic (BP) limb to permit post-RYGB exposure of isolated bowel to sweet taste stimulants. Lean Sprague Dawley (n = 13) and obese Zucker diabetic fatty rats (n = 15) underwent RYGB with BP catheter placement. On postoperative day 11 (POD 11), rats received catheter infusions of saccharin [sweet taste receptor (T1R2/3) agonist] or saline (control). Jejunum was analyzed for changes in glucose transporter/sensor mRNA expression and functional sodium-glucose transporter 1 (SGLT1)-mediated glucose uptake. Saccharin infusion did not alter glucose uptake in the Roux limb of RYGB rats. Intestinal expression of the glucose sensor T1R2 and transporters (SGLT1, glucose transporter 2) was similar in saccharin- vs. saline-infused rats of both strains. However, the abundance of SGLT3b mRNA, a putative glucose sensor, was higher in the common limb vs. BP/Roux limb in both strains of bypassed rats and was significantly decreased in the Roux limb after saccharin infusion. We concluded that failure of BP limb exposure to saccharin to increase Roux limb glucose uptake suggests that isolation of T1R2/3 is unlikely to be involved in metabolic benefits of RYGB, as restimulation failed to reverse changes in intestinal glucose absorption capacity. The altered expression pattern of SGLT3 after RYGB warrants further investigation of its potential involvement in resolution of T2D after RYGB.

Role of growth factors in control of pancreatic beta cell mass: focus on betatrophin.

PURPOSE OF REVIEW: Betatrophin is a newly described hormone, which potently stimulates beta cell replication in mice. This discovery has engendered great hope that it could prove clinically important in the treatment of type 1 and type 2 diabetes. RECENT FINDINGS: Betatrophin, a 198-amino acid protein secreted by liver and adipose tissue, stimulates growth of pancreatic beta cell mass in insulin-resistant mice. Betatrophin has previously been named RIFL, lipasin, and ANGPLT8, and its salutory effects on lipid metabolism have been described in mouse and human studies. Serum betatrophin levels in humans correlate with improved adipose tissue lipid storage and lower serum triglyceride levels in the fed state, but do not correlate with insulin resistance or carbohydrate tolerance in humans. Betatrophin has not yet been shown to have an effect on beta cell replication in human pancreatic islets. SUMMARY: Many endocrine and paracrine factors, of which betatrophin is the newest described, increase beta cell mass in murine models. None of these factors, including betatrophin, have displayed the same activity in clinical studies. This may reflect a profound species difference in beta cell regeneration pathways in mice and humans.

Disrupted circadian rhythmicity of the intestinal glucose transporter SGLT1 in Zucker diabetic fatty rats.

BACKGROUND: Intestinal absorptive capacity shows a circadian rhythm synchronized with eating patterns. Disrupting these coordinated rhythms, e.g., with shift work, may contribute to metabolic disease. Circadian expression of nutrient transporters has not been studied in metabolic disease. We studied the circadian rhythm of intestinal transporter sodium glucose co-transporter type 1 (SGLT1) in an obese diabetic rat. METHODS: We compared obese Zucker diabetic fatty (ZDF) rats to lean ZDF littermates. Temporal feeding patterns were assessed, then rats were harvested at Zeitgeber (ZT, ZT0 = 7:00 a.m.) 3, 9, or 15 to measure insulin resistance, SGLT1 expression and intestinal glucose absorption capacity. Regulators of SGLT1 (sweet taste receptor T1R2/3; clock genes) were measured to elucidate underlying mechanisms. RESULTS: Both groups exhibited altered circadian food intake. Obese ZDF rats lost circadian rhythmicity of SGLT1 mRNA expression and functional activity. Lean ZDF rats maintained rhythmicity of SGLT1 mRNA expression but that of functional glucose absorption was blunted. Circadian rhythms of intestinal clock genes were maintained in both groups. Neither group had discernible rhythms of intestinal GLUT2 (glucose transporter) or T1R2 (sweet taste receptor component) mRNA expression. In summary, lean and obese ZDF rats exhibited similar disruptions in circadian feeding. Glucose intolerance was evident in lean rats, but only obese rats further developed diabetes and exhibited disrupted circadian rhythmicity of both SGLT1 mRNA expression and function. CONCLUSIONS: Our findings suggest that disrupted circadian feeding rhythms contribute to glucose intolerance, but additional factors (genetics, changes in nutrient sensing/transport) are needed to lead to full diabetes.

Upregulation of proapoptotic microRNA mir-125a after massive small bowel resection in rats.

OBJECTIVE: Short bowel syndrome remains a condition of high morbidity and mortality, and current therapeutic options carry significant side effects. To identify new treatments we focused on postresection changes in microRNAs--short noncoding RNAs, which suppress target genes--and suggest a previously undiscovered role for microRNA-125a (mir-125a) in intestinal adaptation. METHODS: Rats underwent either 80% massive small bowel resection or transection and were harvested after 48 hours. Jejunum was harvested for microRNA microarrays, laser capture microdissection, and RNA and protein analysis. Mir-125a was overexpressed in intestinal epithelium-6 (crypt-derived) cells (IEC-6) and effects on proliferation and apoptosis determined using MTS and flow cytometry. Expression of potential targets of mir-125a in rat jejunum and IEC-6 cells was determined using quantitative real-time polymerase chain reaction (RNA) and Western blotting (protein). RESULTS: Resection upregulated mir-125a and mir-214 by 2.4-folds and 3.2-folds, respectively. Highest levels of expression were noted in the crypt fraction. Mir-125a overexpression induced apoptosis and resultant growth arrest in IEC-6 cells. The expression of the prosurvival Bcl-2 family member Mcl-1 was downregulated in both mir-125a-overexpressing IEC-6 cells and in jejunum of resected rats, confirming Mcl-1 as a previously undiscovered target of mir-125a. CONCLUSIONS: Upregulation of mir-125a suppresses the prosurvival protein Mcl1, producing the increase in apoptosis known to accompany the proliferative changes characteristic of intestinal adaptation. Our data highlight a potential role for microRNAs as mediators of the adaptive process and may facilitate the development of new therapeutic options for short bowel syndrome.

PER1 modulates SGLT1 transcription in vitro independent of E-box status.

BACKGROUND AND AIMS: The intestine demonstrates profound circadian rhythmicity in glucose absorption in rodents, mediated entirely by rhythmicity in the transcription, translation, and function of the sodium glucose co-transporter SGLT1 (Slc5a1). Clock genes are rhythmic in the intestine and have been implicated in the regulation of rhythmicity of other intestinal genes; however, their role in the regulation of SGLT1 is unknown. We investigated the effects of one clock gene, PER1, on SGLT1 transcription in vitro. METHODS: Caco-2 cells were stably transfected with knockdown vectors for PER1 and mRNA expression of clock genes and SGLT1 determined using quantitative polymerase chain reaction (qPCR). Chinese hamster ovary (CHO) cells were transiently cotransfected with combinations of the PER1 expression vectors and the wild-type SGLT1-luciferase promoter construct or the promoter with mutated E-box sequences. RESULTS: Knockdown of PER1 increased native SGLT1 expression in Caco-2 enterocytes, while promoter studies confirmed that the inhibitory activity of PER1 on SGLT1 occurs via the proximal 1 kb of the SGLT1 promoter. E-box sites exerted a suppressive effect on the SGLT1 promoter; however, mutation of E-boxes had little effect on the inhibitory activity of PER1 on the SGLT1 promoter suggesting that the actions of PER1 on SGLT1 are independent of E-boxes. CONCLUSIONS: Our findings suggest that PER1 exerts an indirect suppressive effect on SGLT1, possibly acting via other clock-controlled genes binding to non-E-box sites on the SGLT1 promoter. Understanding the regulation of rhythmicity of SGLT1 may lead to new treatments for the modulation of SGLT1 expression in conditions such as malabsorption, diabetes, and obesity.

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.

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.

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.

Novel P2 promoter-derived HNF4alpha isoforms with different N-terminus generated by alternate exon insertion.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a critical transcription factor for pancreas and liver development and functions in islet beta cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4alpha variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4alpha variants designated HNF4alpha10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human=222 nt, rodent=136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4alpha10-alpha12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding alpha7-alpha9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4alpha10-alpha12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4alpha than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4alpha mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

Nonmetabolizable glucose compounds impart cryotolerance to primary rat hepatocytes.

We herein report a novel method for the cryopreservation of hepatocytes using a non-metabolizable glucose derivative in an attempt to mimic the natural cryoprotective adaptations observed in freeze-tolerant frogs. Primary rat hepatocytes were loaded with 3-O-methyl glucose (3OMG) through endogenous glucose transporters without evident toxicity. The 3OMG-loaded hepatocytes were then frozen in a controlled rate freezer down to -80 degrees C and stored in liquid nitrogen at -196 degrees C. Hepatocytes cryopreserved with a relatively small amount of intracellular 3OMG (<0.2 M) showed high post-thaw viability and maintained long-term hepatospecific functions, including synthesis, metabolism, and detoxification. Metabolite uptake and secretion rates were also largely preserved in the cryopreserved hepatocytes. This is the first study to demonstrate the use of the non-metabolizable glucose derivative 3OMG in hepatocyte cryopreservation.

Effect of Portal Glucose Sensing on Systemic Glucose Levels in SD and ZDF Rats.

BACKGROUND: The global epidemic of Type-2-Diabetes (T2D) highlights the need for novel therapeutic targets and agents. Roux-en-Y-Gastric-Bypass (RYGB) is the most effective treatment. Studies investigating the mechanisms of RYGB suggest a role for post-operative changes in portal glucose levels. We investigate the impact of stimulating portal glucose sensors on systemic glucose levels in health and T2D, and evaluated the role of sodium-glucose-cotransporter-3 (SGLT3) as the possible sensor. METHODS: Systemic glucose and hormone responses to portal stimulation were measured. In Sprague-Dawley (SD) rats, post-prandial state was simulated by infusing glucose into the portal vein. The SGLT3 agonist, alpha-methyl-glucopyranoside (αMG), was then added to further stimulate the portal sensor. To elucidate the neural pathway, vagotomy or portal denervation was followed by αMG+glucose co-infusion. The therapeutic potential of portal glucose sensor stimulation was investigated by αMG-only infusion (vs. saline) in SD and Zucker-Diabetic-Fatty (ZDF) rats. Hepatic mRNA expression was also measured. RESULTS: αMG+glucose co-infusion reduced peak systemic glucose (vs. glucose alone), and lowered hepatic G6Pase expression. Portal denervation, but not vagotomy, abolished this effect. αMG-only infusion lowered systemic glucose levels. This glucose-lowering effect was more pronounced in ZDF rats, where portal αMG infusion increased insulin, C-peptide and GIP levels compared to saline infusions. CONCLUSIONS: The portal vein is capable of sensing its glucose levels, and responds by altering hepatic glucose handling. The enhanced effect in T2D, mediated through increased GIP and insulin, highlights a therapeutic target that could be amenable to pharmacological modulation or minimally-invasive surgery.

Foregut exclusion disrupts intestinal glucose sensing and alters portal nutrient and hormonal milieu.

The antidiabetes effects of Roux-en-Y gastric bypass (RYGB) are well-known, but the underlying mechanisms remain unclear. Isolating the proximal small intestine, and in particular its luminal glucose sensors, from the nutrient stream has been proposed as a critical change, but the pathways involved are unclear. In a rodent model, we tested the effects of isolating and then stimulating a segment of proximal intestine using glucose analogs to examine their impact on glucose absorption (Gabsorp) and hormone secretion after a glucose bolus into the distal jejunum. Analogs selective for sodium-glucose cotransporter (SGLT) family members and the sweet taste receptor were tested, and measurements of the portosystemic gradient were used to determine Gabsorp and hormone secretion, including GLP-1. Proximal intestinal isolation reduced Gabsorp and GLP-1 secretion. Stimulation of the glucose-sensing protein SGLT3 increased Gabsorp and GLP-1 secretion. These effects were abolished by vagotomy. Sweet taste receptor stimulation only increased GLP-1 secretion. This study suggests a novel role for SGLT3 in coordinating intestinal function, as reflected by the concomitant modulation of Gabsorp and GLP-1 secretion, with these effects being mediated by the vagus nerve. Our findings provide potential mechanistic insights into foregut exclusion in RYGB and identify SGLT3 as a possible antidiabetes therapeutic target.

Selective deletion of the Hnf1beta (MODY5) gene in beta-cells leads to altered gene expression and defective insulin release.

Hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF1beta (or vHNF1) are closely related transcription factors expressed in liver, kidney, gut, and pancreatic beta-cells. Many HNF1 target genes are involved in carbohydrate metabolism. Human mutations in HNF1alpha or HNF1beta lead to maturity-onset diabetes of the young (MODY3 and MODY5, respectively), and patients present with impaired glucose-stimulated insulin secretion. The underlying defect in MODY5 is not known. Analysis of HNF1beta deficiency in mice has not been possible because HNF1beta null mice die in utero. To examine the role of HNF1beta in glucose homeostasis, viable mice deleted for HNF1beta selectively in beta-cells (beta/H1beta-KO mice) were generated using a Cre-LoxP strategy. beta/H1beta-KO mice had normal growth, fertility, fed or fasted plasma glucose and insulin levels, pancreatic insulin content, and insulin sensitivity. However, beta/H1beta-KO mice exhibited impaired glucose tolerance with reduced insulin secretion compared with wild-type mice but preserved a normal insulin secretory response to arginine. Moreover, beta/H1beta-KO islets had increased HNF1alpha and Pdx-1, decreased HNF4 mRNA levels, and reduced glucose-stimulated insulin release. These results indicate that HNF1beta is involved in regulating the beta-cell transcription factor network and is necessary for glucose sensing or glycolytic signaling.

Circadian clock genes and implications for intestinal nutrient uptake.

There has recently been increasing interest in the phenomenon of circadian rhythmicity. We have used circadian rhythms as a means to understanding the regulation of glucose absorption in the intestine. We and others have previously demonstrated rhythmicity in intestinal glucose uptake, mediated by rhythmicity in the expression of the sodium glucose cotransporter 1. Rhythmicity of clock gene expression was subsequently confirmed in the intestine, a phenomenon also demonstrated in other viscera. Clock genes have since been shown via a combination of in vitro and in vivo techniques to play a role in the transcriptional regulation of key absorptive proteins.

Boosting native promoter activities with non-adjacent response-element multimers.

Effective gene therapy requires regulated gene expression and vector safety. We developed a strategy to exponentially increase native promoter activity while retaining inherent regulation by inserting multi-copy response elements (REs) into non-adjacent locations. For the hepatocyte nuclear factor (HNF) 4α-dependent Hnf1a (MODY3) gene, HNF4α stimulation increased from 5- to 90-fold by inserting 3 additional HNF4α REs (H4REs). Constructing a promoter with two 4xH4REs 0.25kb apart by duplicating the 4xH4RE fragment increased stimulation to >1000-fold. HNF4α-induced protein expression by the duplicate 4xH4RE Hnf1a promoter was comparable to a viral promoter. Converting the two Apolipoprotein C3 (ApoC3) H4REs spaced 0.61kb apart to 4xH4REs achieved a similar result. Increasing spacing to 2.1kb with non-promoter DNA abolished the augmentation. Finally, converting the HNF1α RE of the HNF4A (MODY1) P2 promoter to 4xH1RE and adding a second 4xH1RE 0.84kb upstream increased HNF1α stimulation from 26- to >200-fold. Deleting intervening DNA to produce 0.23-kb spacing increased stimulation to >500-fold. Spaced multi-copy RE motifs is a novel strategy for engineering promoters that boosts activity far beyond other techniques. Augmentation of three promoters suggests that this approach is potentially applicable to other promoters for gene therapy and might obviate the need for viral promoters.

Circadian variation in intestinal dihydropyrimidine dehydrogenase (DPD) expression: a potential mechanism for benefits of 5FU chrono-chemotherapy.

BACKGROUND: 5-fluorouracil (5FU) is associated with significant GI side-effects. Randomized trials have shown a 50% reduction in severe diarrhea with chrono-chemotherapy versus conventional regimens at similar doses. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in 5FU breakdown. We hypothesized that DPD has a circadian expression pattern, accounting for the reduced GI side effects of chrono-modulated 5FU therapy. METHODS: Fifty-one rats were killed at 3-hourly intervals over 24 hours. DPD and thymidylate synthase (TS) mRNA in jejunal and colonic mucosa were measured using qRT-PCR. Cosinor analysis was used for statistical comparison. RESULTS: There was a significant circadian rhythm in the DPD mRNA expression in jejunum (1.7-fold, P < .001) and colon (1.5 fold, P < .01), with a peak expression in early sleep phase, and a trough at mid-wake cycle. TS also followed a circadian rhythm in jejunal mucosa with a peak at early rest phase. CONCLUSION: This rhythm in DPD expression may explain the benefit of chrono-chemotherapy. The peak of DPD expression in sleep phase in rats corresponds to time for lower GI adverse effects in chrono-chemotherapy in human trials. We believe better understanding of this process allows development of novel approaches to optimize the timing of chemotherapy without the administrative challenges of chronotherapy.

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.

Function of HNF1 in the pathogenesis of diabetes.

The importance of hepatocyte nuclear factors (HNFs), as well as other transcription factors in ß-cell development and function, was underlined by the characterization of human mutations causing maturity-onset diabetes of the young (MODY). HNF1A and HNF1B mutations lead to MODY forms 3 and 5, respectively. Thus, transcriptional control is an essential mechanism underlying the precise metabolic control exerted by ß-cells in regulating insulin release. The diabetes phenotype of MODY3 (HNF1α) and the phenotypes of MODY5 (HNF1ß), which can also include renal disease and genitourinary malformations, as well as neonatal diabetes and pancreatic agenesis, have now been described. However, detailed molecular pathology remains elusive. The large array of dominant-negative and deletion mutations, and the lack of structure-phenotype relationships for most mutations, have not helped us to formulate a mechanistic understanding. Further molecular studies of HNF1 actions and gene regulation are anticipated to provide useful insights into ß-cell biology and potential therapeutic tools.