Customization of biliopancreatic limb length to modulate and sustain antidiabetic effect of gastric bypass surgery.

Although Roux-en-Y Gastric Bypass (RYGB) remains the most effective treatment for obesity and type 2 diabetes (T2D), many patients fail to achieve remission, or relapse. Increasing intestinal limb lengths of RYGB may improve outcomes, but the mechanistic basis for this remains unclear. We hypothesize biliopancreatic (BP) limb length modulates the antidiabetic effect of RYGB. Rats underwent RYGB with a 20-cm (RYGB-20cm) or 40-cm (RYGB-40cm) BP limb and were compared with control animals. After 2 and 4 wk, portal and systemic blood was sampled during intestinal glucose infusion. Portosystemic gradient was used to calculate intestinal glucose utilization (Gutil), absorption (Gabsorp), and hormone secretion. Intestinal morphology and gene expression were assessed. At 2 wk, Gabsorp progressively decreased with increasing BP limb length; this pattern persisted at 4 wk. Gutil increased ≈70% in both RYGB-20cm and -40cm groups at 2 wk. At 4 wk, Gutil progressively increased with limb length. Furthermore, Roux limb weight, and expression of hexokinase and preproglucagon, exhibited a similar progressive increase. At 4 wk, glucagon-like peptide-1 and -2 levels were higher after RYGB-40cm, with associated increased secretion. We conclude that BP limb length modulates multiple antidiabetic mechanisms, analogous to the dose-response relationship of a drug. Early postoperatively, a longer BP limb reduces Gabsorp. Later, Gutil, Roux limb hypertrophy, hormone secretion, and hormone levels are increased with longer BP limb. Sustained high incretin levels may prevent weight regain and T2D relapse. These data provide the basis for customizing BP limb length according to patient characteristics and desired metabolic effect. NEW & NOTEWORTHY Biliopancreatic limb length in gastric bypass modulates multiple antidiabetic mechanisms, analogous to the dose-response relationship of a drug. With a longer biliopancreatic limb, Roux limb hypertrophy, increased glucose utilization, reduced glucose absorption, and sustained high incretin levels may prevent weight regain and diabetes relapse.

Relative contributions of afferent vagal fibers to resistance to diet-induced obesity.

BACKGROUND: We previously demonstrated vagal neural pathways, specifically subdiaphragmatic afferent fibers, regulate expression of the intestinal sodium-glucose cotransporter SGLT1, the intestinal transporter responsible for absorption of dietary glucose. We hypothesized targeting this pathway could be a novel therapy for obesity. We therefore tested the impact of disrupting vagal signaling by total vagotomy or selective vagal de-afferentation on weight gain and fat content in diet-induced obese rats. METHODS: Male Sprague-Dawley rats (n = 5-8) underwent truncal vagotomy, selective vagal de-afferentation with capsaicin, or sham procedure. Animals were maintained for 11 months on a high-caloric Western diet. Abdominal visceral fat content was assessed by magnetic resonance imaging together with weight of fat pads at harvest. Glucose homeostasis was assessed by fasting blood glucose and HbA1C. Jejunal SGLT1 gene expression was assessed by qPCR and immunoblotting and function by glucose uptake in everted jejunal sleeves. RESULTS: At 11-months, vagotomized rats weighed 19% less (P = 0.003) and de-afferented rats 7% less (P = 0.19) than shams. Vagotomized and de-afferented animals had 52% (P < 0.0001) and 18% reduction (P = 0.039) in visceral abdominal fat, respectively. There were no changes in blood glucose or glycemic indexes. SGLT1 mRNA, protein and function were unchanged across all cohorts at 11-months postoperatively. CONCLUSIONS: Truncal vagotomy led to significant reductions in both diet-induced weight gain and visceral abdominal fat deposition. Vagal de-afferentation led to a more modest, but clinically and statistically significant, reduction in visceral abdominal fat. As increased visceral abdominal fat is associated with excess morbidity and mortality, vagal de-afferentation may be a useful adjunct in bariatric surgery.

Diurnal rhythmicity in intestinal SGLT-1 function, V(max), and mRNA expression topography.

Mechanisms underlying the circadian rhythmicity in intestinal sugar absorption remain unclear. To test whether this rhythmicity is caused by changes in Na(+)-glucose cotransporter 1 (SGLT-1) function, we measured phloridzin-inhibitable sugar fluxes as an index of SGLT-1 activity. Jejunum obtained from rats killed at 6-h intervals during a 12-h light-dark cycle (CT0 is circadian time 0 h, time of light onset) were mounted in Ussing chambers, and 3-O-methylglucose (3-OMG) fluxes were calculated before and after addition of phloridzin. 3-OMG-induced change in short-circuit current and absorptive flux were significantly greater at CT9 than at CT3. This increase was phloridzin inhibitable. Kinetic studies indicated a significant increase in SGLT-1 maximal velocity (V(max)) at CT9. Food intake between CT3 and CT9 was <10% of the daily total, indicating that the increased SGLT-1 activity was anticipatory. Diurnicity of SGLT-1 mRNA was confirmed by Northern blotting. Expression topography analyzed by in situ hybridization revealed more intense labeling along the entire villus axis at CT9 and CT15 compared with CT3 and CT21. We conclude that diurnicity in intestinal sugar absorption is caused by periodicity in SGLT-1 V(max).

Circadian periodicity of intestinal Na+/glucose cotransporter 1 mRNA levels is transcriptionally regulated.

Intestinal expression of the high affinity Na+/glucose cotransporter 1 (SGLT1), which absorbs dietary glucose and galactose, exhibits both circadian periodicity in its activity and induction by dietary carbohydrate. Because the daily variation in SGLT1 activity is established by the feeding schedule (whether ad libitum or imposed) and persists in the absence of food, this variation has been described as anticipatory. To delineate the mechanisms regulating SGLT1, its expression was examined in rats maintained in a 12-h photoperiod with free access to chow. SGLT1 mRNA levels varied significantly, with the maximum abundance occurring near the onset of dark and the minimum near the onset of light. The SGLT1 transcription rate was 7-fold higher in the morning (1000-1100 h) than in the afternoon (1600-1700 h). An element for hepatocyte nuclear factor 1 (HNF-1) was identified in the SGLT1 promoter that formed different complexes with small intestinal nuclear extracts, depending on the time when the source animal was killed. Serological tests indicated that HNF-1alpha was present in complexes throughout the day, while HNF-1beta binding exhibited circadian periodicity. We propose that exchange of HNF-1 dimerization partners contributes to circadian changes in SGLT1 transcription. Because SGLT1 mRNA levels also varied in rhesus monkeys (offset by approximately one-half day from rats), a similar mechanism appears to be present in primates.

Glucose regulation of glucose transporters in cultured adult and fetal hepatocytes.

GLUT2 is the major glucose transporter of adult hepatocytes. In vivo, membrane GLUT1 is localized to a ring of perivenous cells and increases slightly after fasting or insulin deprivation. GLUT1 also increases in vitro after prolonged culture of isolated adult hepatocytes. We have previously shown that GLUT1 mRNA, protein, and activity are present in the rat fetal hepatocyte, and that both GLUT1 and GLUT2 are important for the pattern of glucose transport in the fetal hepatocyte. We tested the hypothesis that the hypothesis that the postnatal increase in circulating glucose is one of the regulators of the changed pattern of GLUT1 and GLUT2 in the hepatocyte after the fetal to neonatal transition. Fetal and adult rat hepatocytes were cultured for 45 hours in supplemented Dulbecco's modified Eagle's medium at glucose concentrations of 1, 8.3, or 30 mmol/L. Culture at 8.3 and 30 mmol/L glucose diminished GLUT1 mRNA levels were lower in adult versus fetal hepatocyte cultures at 8.3 and 30 mmol/L (P < .05). Similarly, GLUT1 protein levels were significantly diminished in hepatocytes cultured at higher medium glucose (P < .05 for fetal cells at 30 v 1 mmol/L; P < .05 for adult cells at 8.3 and 30 v 1 mmol/L). GLUT2 mRNA abundance was enhanced by medium glucose in adult hepatocytes (P < .05 at 8.3 and 30 v 1 mmol/L) and was unchanged by medium glucose in fetal hepatocytes. In contrast, GLUT2 protein level was unchanged by medium glucose in adult hepatocytes, and was diminished at 30 mmol/L as compared with mmol/L glucose in fetal hepatocytes (P < .05). In confirmation of these findings, uptake of 2-deoxyglucose (2-DOG) by fetal hepatocytes was significantly diminished after culture in 8.3 or 30 mmol/L glucose versus 1 mmol/L glucose (P < .05 and < .01, respectively). These studies confirm that the fetal hepatocyte glucose transporter pattern could be maintained in part by low fetal portal glucose levels. However, the resistance of the fetal hepatocyte glucose transporter pattern as compared with that of the adult hepatocyte to the effects of hyperglycemia suggests additional undefined control mechanisms.

Differentiation-dependent expression of the Na+/glucose cotransporter (SGLT1) in LLC-PK1 cells: role of protein kinase C activation and ongoing transcription.

We examined changes in the mRNA level of SGLT1, a Na+/glucose cotransporter, by the differentiation status of LLC-PK1 renal epithelial cells. Proliferating (undifferentiated) cells revealed no detectable SGLT1 mRNA by Northern blot analysis. However, when cells became confluent and differentiated into polarized monolayers, there was an abrupt appearance of the SGLT1 mRNA. When confluent (differentiated) cells were dedifferentiated by reseeding at a subconfluent density, SGLT1 mRNA levels decreased quickly to nondetectable levels (t1/2 = 1.5 h), while the mRNA levels of gamma-glutamyltranspeptidase, another differentiation marker, decreased only slowly (t1/2 > 40 h). This decrease in SGLT1 mRNA was completely blocked by H-7, a protein kinase inhibitor. Since protein kinase C was highly activated in the undifferentiated cells and treatment of differentiated cells with a phorbol ester also induced quick and complete loss of SGLT1 mRNA (t1/2 = 1.5 h) but not of gamma-glutamyltranspeptidase mRNA, protein kinase C activation appears to be involved in the dedifferentiation-induced decrease in SGLT1 mRNA. Although the phorbol ester-induced decrease in the SGLT1 mRNA level was blocked completely by inhibition of transcription, inhibitors of translation blocked the decrease in mRNA levels only partially.

GLUT-1 and GLUT-2 mRNA, protein, and glucose transporter activity in cultured fetal and adult hepatocytes.

To understand glycogenesis in the fetal hepatocyte, we examined glucose transport in cultured fetal and adult male rat hepatocytes. GLUT-1 mRNA was detected in fetal hepatocytes at isolation but in adult hepatocytes only after culture. GLUT-1 mRNA was more abundant in fetal than in adult hepatocytes (P < 0.005). GLUT-1 protein paralleled its message. GLUT-2 mRNA was more abundant in adult than in fetal hepatocytes (P < 0.05), and abundance did not change during culture, but GLUT-2 protein was discordantly regulated. There was more GLUT-2 protein in fetal hepatocytes at 45 h (P < 0.025). An Eadie-Hofstee plot of 3-O-methylglucose transport appeared to have two linear components. One component was presumed to be GLUT-1 [variable Michaelis constant (Km) approximating 6-8 mM, maximal uptake rate (Vmax) for fetal vs. adult hepatocytes 106 vs. 35 nmol.min-1.mg protein-1], and a second was presumed to be GLUT-2 (Km of 23 mM, Vmax for fetal vs. adult hepatocytes 198 vs. 92 nmol.min-1.mg protein-1). Early phosphorylation of 2-deoxyglucose was greater in fetal than in adult hepatocytes, but transport was always greater than phosphorylation. Increased expression of both GLUT-1 and GLUT-2 by fetal hepatocytes permits greater glucose uptake and positions the fetal rat hepatocyte for efficient glycogenesis at low plasma glucose concentration.

Increased expression of eukaryotic translation initiation factors eIF-4E and eIF-2 alpha in response to growth induction by c-myc.

Although activation of c-myc is a critical step in the development of lymphomas and other tumors, its normal function(s) in cell growth remain obscure because few myc-regulated genes are known. myc expression normally increases in response to mitogens and peaks in G1 when additional protein synthesis is required for cell-cycle progression. Protein synthesis is controlled by the availability of translation initiation factors, including the mRNA cap binding protein (eIF-4E) and the alpha subunit of the eIF-2 complex that binds the initiator Met-tRNA. Consequently we examined eIF-4E and eIF-2 alpha for evidence of regulation by c-myc. Expression of eIF-4E and eIF-2 alpha correlated with c-myc expression in fibroblasts after growth stimulation. In addition, expression of eIF-4E and eIF-2 alpha was increased in myc-transformed rat embryo fibroblasts but was not increased in ras-transformed cells. Transcription rates of eIF-4E and eIF-2 alpha mRNAs were regulated by c-myc in cells expressing an estrogen receptor-Myc fusion protein. Finally, electrophoretic mobility-shift assays identified a sequence element in the eIF-2 alpha promoter, TCCGCAT-GCGCG, which was specifically retarded by extracts of myc-expressing cells. c-myc is thought to deregulate the growth of cancer cells by activating transcription, suggesting that specific genes regulated by c-myc should also function as oncogenes. In previous studies these translation initiation factors could induce neoplastic growth because overexpression of eIF-4E-transformed cells and inhibition of a suppressor of eIF-2 alpha (eIF-2 alpha kinase) also caused malignant transformation. Our studies suggest that one important biological function of c-myc may be to increase cell growth by increasing expression of eIF-4E and eIF-2 alpha.

Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells.

Hepatocyte growth factor (HGF) is a potent mitogen for primary hepatocytes. Therefore, we examined HGF as a possible autocrine growth factor in hepatocellular carcinoma (HCC). We introduced an albumin-HGF expression vector into Fao HCC cells and transgenic mice. Expression of the albumin-HGF vector in Fao HCC cells inhibited their growth in vitro. In vivo, FaoHGF cells produced tumors that averaged 10% of the sizes of G418-resistant controls when transplanted into nude mice. In contrast, hepatocytes from transgenic mice expressing HGF grew more rapidly than did those from normal siblings. Further, growth of eight additional HCC cell lines was inhibited by the addition of recombinant HGF. Finally, of 35 tumor cell lines surveyed, only 6 cell lines expressed HGF mRNA, and no HCC cell line expressed HGF. Although HGF stimulates normal hepatocytes, it is a negative growth regulator for HCC cells.

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.

Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (ii) in vivo, when rats were subjected to starvation for greater than or equal to 2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment.

Sodium butyrate increases glucose transporter expression in LLC-PK1 cells.

The effect of sodium butyrate on the expression of the facilitated glucose transporter (GT) was investigated in the pig kidney cell line LLC-PK1. When cells were treated with butyrate, GT mRNA expression was remarkably enhanced with a maximal effect at 5 mM. Levels of GT mRNA were increased at 1 day after butyrate treatment and continued to increase for at least 4 days; however, acetate and propionate did not affect GT mRNA levels significantly. The induction of GT mRNA by butyrate was accompanied by an increase in GT function. The expression of GT mRNA decreased in HepG2, HT-29, and COS cells by treatment with butyrate for 1 day. Interestingly, glucose deprivation of LLC-PK1 cells reduced the induction of GT mRNA by butyrate, although starvation itself slightly enhanced steady-state GT mRNA levels. Therefore, expression of GT in LLC-PK1 cells is strongly induced by butyrate by a pathway that apparently depends on the presence of glucose in culture medium.

Partial suppression of anchorage-independent growth and tumorigenicity in immunodeficient mice by transfection of the H-2 class I gene H-2Ld into a human colon cancer cell line (HCT).

Many human tumors, particularly those of epithelial origin, appear to express greatly reduced levels of major histocompatibility complex class I antigens on their surface. It has been previously reported that the class I gene H-2Ld, introduced into adenovirus type 12-transformed mouse cells, induces reversal of oncogenesis in immunocompetent BALB/c mice. We have tested the hypothesis that the H-2Ld gene, when transfected into HCT colon cancer cells, may alter their transformed phenotype. Two H-2Ld transfectants, HCT-Ii and HCT-If, were found to exhibit a markedly reduced-to-virtually suppressed ability to form colonies in soft agar in comparison to a transfectant (HCTh) carrying only the neomycin-resistance gene. We also compared the tumorigenicity of HCTh vs. HCT-If cells in two different strains of immunodeficient mice: nude (T-) and triple-deficient mutants (T-, NK-, B-). At 28 days postinjection of 10(7) and 10(6) cells, the size and growth rate of HCT-If tumors were greatly reduced compared to HCTh cells. Therefore, as assayed in immunodeficient animals, expression of the class I H-2Ld gene in HCT cells appears to correlate with partial suppression of the tumorigenic phenotype, suggesting that the expression of a transfected class I gene may by itself alter the phenotype of the recipient cell and that such phenotypic changes may be independent of the immune system.

Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energized membrane and not simply from ATP. Treatment of the vesicles with protease, under conditions that did not interfere with subsequent protein synthesis, also inactivated them for subsequent translocation. We conclude that export of some proteins requires protein-containing machinery in the cytoplasmic membrane that derives energy from the proton motive force.

Osmotic control of kdp operon expression in Escherichia coli.

Turgor pressure, the difference in osmotic pressure across the inner membrane, has been found to regulate expression of the kdp operon in Escherichia coli. The kdp operon codes for a high-affinity repressible transport system for the uptake of potassium. We have studied the regulation of Kdp expression in a strain in which the gene for beta-galactosidase, lacZ, was placed under control of the kdp promotor. Neither internal nor external K+ concentrations directly controlled Kdp expression. Only when the external K+ concentration was reduced to the point of limiting growth was the kdp operon expressed. An increase in external osmolarity at constant K+ concentration, a procedure that reduces turgor pressure, caused expression of the kdp operon. As the magnitude of the osmotic shift was increased, corresponding to greater decreases in turgor pressure, the amount of Kdp expression also increased. The kdp operon thus appears to be controlled by changes in a physical force, the turgor pressure.

Cation transport in Escherichia coli. IX. Regulation of K transport.

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.

Functional organization of the kdp genes of Escherichia coli K-12.

The kdp genes code for a high-affinity and repressible K+ transport system. The regulation and organization of the kdp genes were analyzed by studies of constitutive mutants and of strains in which bacteriophage lambda is integrated into the kdp genes. The polar effects of lambda integration demonstrate that three of the kdp genes form an operon, kdpABC, read from A to C. The kdpD gene is a separate transcription unit and is the site of mutations making expression of the kdp genes partially constitutive. The constitutive mutants are dominant to kdpD+ in diploids. These findings, the fact that kdpD mutations identified previously are Kdp-, and the existence of intracistronic complementation between some kdpD mutations indicate that the kdpD gene product is an oligomeric positive regulator of the kdp genes. Deletions extending clockwise from kdp as far as the gltA locus were isolated from strains with bacteriophage lambda integrated into kdpD. Plaque-forming transducing lambda phages carrying the kdpABC operon were isolated.