abstract: Gastric bypass surgery leads to marked improvements in glucose tolerance and insulin sensitivity in obese type 2 diabetes (T2D); the impact on glucose fluxes in response to a physiological stimulus, such as a mixed meal test (MTT), has not been determined. We administered an MTT to 12 obese T2D patients and 15 obese nondiabetic (ND) subjects before and 1 year after surgery (10 T2D and 11 ND) using the double-tracer technique and modeling of β-cell function. In both groups postsurgery, tracer-derived appearance of oral glucose was biphasic, a rapid increase followed by a sharp drop, a pattern that was mirrored by postprandial glucose levels and insulin secretion. In diabetic patients, surgery lowered fasting and postprandial glucose levels, peripheral insulin sensitivity increased in proportion to weight loss (∼30%), and β-cell glucose sensitivity doubled but did not normalize (compared with 21 nonsurgical obese and lean controls). Endogenous glucose production, however, was less suppressed during the MMT as the combined result of a relative hyperglucagonemia and the rapid fall in plasma glucose and insulin levels. We conclude that in T2D, bypass surgery changes the postprandial response to a dumping-like pattern and improves glucose tolerance, β-cell function, and peripheral insulin sensitivity but worsens endogenous glucose output in response to a physiological stimulus.
author: Stefania Camastra; Elza Muscelli; Amalia Gastaldelli; Jens J. Holst; Brenno Astiarraga; Simona Baldi; Monica Nannipieri; Demetrio Ciociaro; Marco Anselmino; Andrea Mari; Ele FerranniniCorresponding author: Stefania Camastra, <firstname.lastname@example.com>.
date: 2013-11
references:
title: Long-Term Effects of Bariatric Surgery on Meal Disposal and β-Cell Function in Diabetic and Nondiabetic Patients

Mounting evidence supports bariatric surgery as a powerful intervention to induce remission in patients with type 2 diabetes (T2D) (1,2) and to prevent or delay incident T2D (3). This has engendered enthusiasm for bariatric surgery as a treatment for T2D (4) and has encouraged a broadening of the BMI range as an indication for surgery in diabetic patients (5).

Although weight loss and, in the early postoperative period, caloric deficit certainly make a contribution to improve glucose tolerance, surgery itself may trigger weight-independent mechanisms eventually translating into favorable metabolic effects. This postulate is based on early animal studies (6) and, in humans, on evidence that metabolic changes sometimes precede sizeable weight loss or are disproportionate to the amount of weight lost (7). In this regard, there is evidence that different bariatric procedures (e.g., Roux-en-Y gastric bypass \[RYGB\], biliopancreatic diversion, and sleeve gastrectomy) may engage putative weight-independent mechanisms to different extents or involve altogether different mechanisms (8,9).

A number of previous studies have documented the effects of the most popular bariatric operation, RYGB, on glycemic control and incretin hormones (10–24), and mechanistic studies have explored the ability of RYGB to enhance insulin action and β-cell function. The great majority of these studies have used methods based on fasting measurements (e.g., homeostasis model assessments), oral glucose tolerance test–based surrogate indices of insulin sensitivity and β-cell function, or euglycemic-hyperinsulinemic clamp settings (14,16–21). A recent study (24) has taken a more physiological approach by comparing the impact of RYGB and gastric banding on the disposition of a mixed meal, with the use of a double-tracer technique, in nondiabetic (ND) subjects studied before and shortly after the operation (∼20 weeks). In the current study of morbidly obese patients with T2D, we aimed at measuring the impact of RYGB on chief physiologic determinants of meal disposal long after surgery (when body weight and metabolic adaptation have stabilized) and assessing their relation to weight loss and the attendant changes in the hormonal milieu.

# RESEARCH DESIGN AND METHODS

## Subjects.

We studied 12 morbidly obese patients with T2D and 15 sex- and BMI-matched morbidly obese ND patients. Diabetes was newly diagnosed in three patients, whereas in the other nine patients, diabetes duration was 3.9 ± 1.2 years (range 1–10). HbA~1c~ was 7.2 ± 0.4% (55 ± 5 mmol/mol); six patients were being treated by diet alone and six by oral hypoglycemic agents (three by metformin alone and three by metformin plus a sulfonylurea). Antidiabetic medication was discontinued 1 week before the metabolic studies. These 27 subjects all underwent laparoscopic RYGB; 10 T2D and 11 ND patients were restudied 1 year after surgery. Two control groups were included, consisting of 7 lean healthy volunteers and 14 obese ND volunteers whose BMI was matched to that of the RYGB patients at 1 year postsurgery. Thus, a total of 69 complete metabolic studies were performed.

This study was approved by the local ethics committee. The nature and purpose of the study were carefully explained to all participants before they provided written consent to participate.

## Study design.

At baseline and follow-up, subjects received a mixed meal test (MTT) with a double-tracer protocol. In brief, after an overnight (12-h) fast, subjects were admitted to our Clinical Research Unit at 8:00 a.m., and a polyethylene cannula was inserted into an antecubital vein for the infusion of all test substances. A second catheter was inserted retrogradely into an ipsilateral wrist vein on the dorsum of the hand for blood sampling, and the hand was kept in a heated box at 65°C to achieve arterialization of venous blood. Baseline blood samples were drawn to measure plasma glucose, insulin, C-peptide, glucagon, glucagon-like peptide 1 (GLP-1), and gastric inhibitory polypeptide (GIP) concentrations and tracer enrichments. The MTT consisted of 75 g glucose in 150 mL water, 40 g parmesan cheese, and one 50-g egg (509 kcal; 16% protein, 28% fat, and 56% carbohydrate). The glucose solution was drunk after the solid component of the meal was consumed over a period of 10 min. The oral glucose drink was enriched with 1-\[^2^H\]glucose in order to trace glucose absorption. A primed (28 µmol ⋅ kg^−1^ × \[fasting plasma glucose\]/5) continuous (0.28 µmol ⋅ min^−1^ ⋅ kg^−1^) infusion of 6,6-\[^2^H~2~\]glucose was started (at time –120 min, or at –180 min in the T2D patients) via the antecubital vein catheter and continued until the end of the study. During the last 20 min of the basal equilibration period (at times –20, –10, and 0 min), blood samples were obtained for the determination of plasma glucose and hormone concentrations and tracer enrichments (when isotopic steady state was achieved). After the basal equilibration period, the meal was consumed over ∼10 min. Plasma samples for the determination of plasma glucose and hormone concentrations and glucose tracer enrichments were obtained at 15, 30, 45, 60, 90, 120, 150, 180, 240, and 300 min after meal ingestion.

## Surgical procedure.

In subjects undergoing laparoscopic RYGB, a small proximal gastric pouch of 30 mL was created with several firings of a linear stapling endocutter; the jejunum was divided 120 cm distal to the ligament of Treitz, and a 2-cm end-to-side gastrojejunostomy was performed by using a hand-sewn technique. A side-to-side jejuno-jejunostomy was then created, 150 cm distal to the gastrojejunostomy (17).

## Analytical procedures.

Plasma glucose was measured by the glucose-oxidase technique (Analox GM-9) and plasma insulin and C-peptide by electrochemiluminescence (on a COBAS e411 instrument; Roche, Indianapolis, IN). Plasma triglycerides and serum HDL cholesterol were assayed in duplicate by standard spectrophotometric methods on a Synchron Clinical System CX4 (Beckman Instruments, Fullerton, CA). 6,6-\[^2^H~2~\]glucose and 1-\[^2^H\]glucose enrichments were measured by gas chromatography–mass spectrometry as described previously (25). Plasma glucagon was assayed by radioimmunoassay (Millipore Corporation, Billerica, MA). Plasma GLP-1 and GIP were assayed by radioimmunoassay as described previously (26). No significant cross-reactivity was declared by kit manufacturers. The sensitivity ranges are 0.003 nmol/L and 1.39 pmol/L for the C-peptide and insulin assay, respectively, 18.5 pg/mL for glucagon, and 1.5 pmol/L for GLP-1.

## Data analysis.

Fat-free mass was estimated with the use of electric bioimpedance on a Tanita scale; of note, this method has been validated in very obese individuals against the deuterated water technique (27). Endogenous glucose production (EGP) in the fasting state and throughout the absorptive period and rate of appearance of the oral glucose component (RaO) of the meal were calculated from the time course of the plasma tracer/tracee ratio of 6,6-\[^2^H~2~\]glucose and 1-\[^2^H\]glucose by two-compartment modeling, as previously described (25). Insulin sensitivity was calculated as the mean metabolic clearance rate of glucose (MCR~G~) during the 5 h of the MTT (from 6,6-\[^2^H~2~\]glucose kinetics) divided by the mean plasma insulin concentration over the same time interval.

β-Cell function was quantitated by mathematical modeling of the plasma C-peptide response, as previously described (28). In brief, the model consists of three blocks: *1*) a model for fitting the glucose concentration profile, the purpose of which is to smooth and interpolate plasma glucose concentrations; *2*) a model describing the dependence of insulin (or C-peptide) secretion on glucose concentration; and *3*) a model of C-peptide kinetics, i.e., the two-exponential model proposed by Van Cauter et al. (29), in which the model parameters are individually adjusted to the subject's anthropometric data. The main parameter of the model is β-cell glucose sensitivity (β-GS), which is calculated as the mean slope of the dose-response function (i.e., relationship between insulin secretion rates \[ISRs\] and plasma glucose concentrations during corresponding times of the MTT). The model also yields an estimate of glucose rate sensitivity, which is the insulin secretory response to the rate of change in plasma glucose concentrations, and total insulin output, which is the total amount of insulin released over the 5 h of the meal (28). Plasma insulin clearance (MCR~I~) was estimated as the ratio of fasting ISR to the fasting plasma insulin concentration.

The prehepatic insulin-to-glucagon molar concentration ratio was estimated by the following formula: {ISR\[*t*\]/hPF + \[I(*t*)\]}/{\[Glg(t)\] × \[1 + MCR~Glg~/hPF\]}, where ISR(*t*) is the ISR at time *t*, hPF is hepatic plasma flow, \[I(*t*)\] and \[Glg(*t*)\] are the measured peripheral plasma concentrations of insulin and glucagon at time *t*, and MCR~Glg~ is the metabolic clearance rate of glucagon. hPF was estimated by multiplying the cardiac index (3.2 L ⋅ min^–1^ ⋅ m^−2^) (30) by a plasma-to-blood ratio of 0.6, and by assuming that hepatic blood flow is 30% of cardiac index (=0.576 L ⋅ min^−1^ ⋅ m^−2^) (31). MCR~Glg~ was taken to be 0.537 L ⋅ min^−1^ ⋅ m^−2^ (32).

The product of mean EGP and mean plasma insulin levels during the meal was taken as an index of hepatic insulin resistance. Areas under time-concentration curves (AUCs) were calculated by the trapezium rule.

## Statistical analysis.

All data are given as the mean ± SEM; parameters that were nonnormally distributed are presented as the median (interquartile range). Mann-Whitney test was used to compare group values, whereas surgery-induced changes were tested by the Wilcoxon signed rank test. Time series were analyzed by ANOVA for repeated measures; for these tests, variables with skewed distribution were log transformed. Group differences over time series were analyzed by two-way ANOVA for repeated measures. Simple associations were tested by calculating the Spearman rank correlation coefficient (*ρ*). Statistical analyses were performed using JMP7.0 and StatView5.0. *P* ≤ 0.05 was considered statistically significant.

# RESULTS

## Baseline.

The four subject groups were matched for sex and age; T2D patients were older and had moderate fasting hyperglycemia (Table 1). After meal ingestion, plasma glucose excursions were similar in lean and obese controls and ND surgical patients, whereas T2D patients showed marked hyperglycemia (Fig. 1A). Fasting and postprandial insulin concentrations were higher in all three obese groups than in lean controls (Fig. 1B and Table 2). Fasting ISR generally increased with obesity as did the total insulin output over the 5 h of the test (Fig. 1C and Table 2). In T2D patients, total insulin output was not different from that of lean controls despite the hyperglycemia. Consequently, β-GS was markedly impaired in this group (Fig. 1D and Table 2).

Anthropometric and metabolic parameters

![](3709tbl1)

Glucose metabolism and insulin secretion

![](3709tbl2)

On the MTT, the RaO was similar in all groups, both in time course (Fig. 1E) and amount (averaging 51, 52, 50, and 62 g over 5 h in lean control subjects, obese control subjects, ND surgical patients, and T2D surgical patients, respectively) (Table 2). In contrast, fasting EGP was higher in each obese group (700 \[277\], 771 \[242\], and 764 \[395\] µmol/min) than in lean controls (580 \[51\] µmol ⋅ min^−1^) and remained higher during the MTT despite the postmeal hyperinsulinemia (AUC~EGP~ = 79 \[46\], 100 \[56\], and 100 \[97\] mmol vs. 61 \[15\] of controls). The difference in EGP time course between each of the obese groups and lean controls (confirmed by repeated-measures ANOVA, *P* \< 0.001 for all) was evident after the initial nadir (Fig. 1F). In the whole dataset, both fasting EGP and AUC~EGP~ were positively related to BMI (*r* = 0.48, *P* = 0.001, and *r* = 0.30, *P* = 0.05, respectively). Plasma glucose clearance rose during the MTT in all groups; its mean value over 5 h was reduced in both surgical groups, especially in T2D patients, in comparison with lean controls despite the hyperinsulinemia (Table 2). As a consequence, insulin sensitivity, as the insulin-mediated MCR~G~, was lower in both obese groups and severely impaired in T2D patients (Table 2).

In all obese groups, fasting plasma glucagon was increased, whereas the meal-induced glucagon increments were blunted. Fasting GLP-1 and GIP levels were increased only in T2D patients, as was AUC~GIP~ (Table 2).

## After surgery.

At 1 year, ND and T2D patients had lost 31 and 35% of their initial body weight, respectively, in an approximate ratio of 1:3 between fat-free mass and fat mass (Table 1). In T2D patients, HbA~1c~ had dropped by an average 1.5%, and all patients were off antidiabetic drug treatment; of them, two patients had impaired glucose tolerance and one of these two also had impaired fasting glucose (5.9 mmol/L). Four patients experienced dumping-like symptoms (tachycardia, sweating, nausea, and diarrhea) after meal ingestion.

On the MTT, the postsurgery plasma glucose and insulin profiles were grossly altered in both ND and T2D subjects, with a large excursion peaking at 60 min followed by a sharp drop to basal levels or below (Fig. 2). In the T2D group, the glucose and insulin AUCs were significantly smaller than preoperatively (Table 2). The time course of insulin secretion ran parallel to that of plasma glucose (Fig. 3); fasting insulin secretion decreased in both groups and total insulin output decreased in the ND group (Table 2). When viewing insulin secretion in the context of the corresponding plasma glucose levels, β-GS was significantly reduced in the ND group postsurgery (although still within the range of the obese control group), and ∼100% increased in T2D patients (although still largely below control values) (Fig. 3). The small decline in β-GS in ND subjects could be due to chronically reduced carbohydrate intake or could simply be time related. Insulin sensitivity improved significantly in both groups (by two-way ANOVA from repeated measures, *F* = 10.5, *P* \< 0.005) to levels similar to those of the obese controls. The estimates of insulin sensitivity fell along the overall relationship describing the association of insulin resistance with BMI, and the surgically induced changes in insulin sensitivity were correlated with the corresponding changes in body weight (Table 2 and Fig. 4).

In both ND and T2D subjects postsurgery, the rate of appearance of oral glucose was similar in total amount (Table 2) but distorted in time course, in a manner resembling the plasma glucose curves closely (Fig. 5). Thus, most oral glucose was absorbed during the first hour after meal ingestion, as the AUC calculated in the first 60 min showed (AUC pre- vs. postsurgery: 122 \[63\] vs. 186 \[37\] mmol in ND and 129 \[81\] vs. 190 \[94\] mmol in T2D, *P* \< 0.04 vs. postsurgery for both). EGP was unchanged in ND as well as T2D patients in the fasting state but rebounded significantly above presurgery values during meal absorption, particularly over the second half of the postcibal period when plasma glucose concentrations were back to basal or below. The EGP time course was increased in both groups (with no significant difference between T2D and ND patients, *P* = 0.74), whether expressed in absolute terms (Fig. 5) or normalized by fat-free mass (*F* = 133.9, *P* \< 0.0001) (Table 2).

Fasting glucagon levels dropped in both ND and T2D, but the response to the meal (as the incremental AUC) was greatly enhanced in both groups (Table 2 and Fig. 6). After surgery, the estimated prehepatic insulin-to-glucagon molar concentration ratio was higher than presurgery (especially in T2D patients) for the first 80–100 min but then fell below preoperative levels for the remainder of the MTT in both groups ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0321/-/DC1)).

The GLP-1, but not the GIP, response was increased in ND as well as T2D; the time profile of the GLP-1 and GIP responses showed a sharper early rise and a rapid drop thereafter (Fig. 6).

MCR~I~, calculated as the ratio of fasting insulin output to fasting peripheral plasma insulin concentrations, was slightly reduced in the obese groups as compared with the lean controls and was significantly increased postsurgery in both ND and T2D (two-way ANOVA).

# DISCUSSION

Before the operation, the metabolic response to the mixed meal in the ND obese subjects was characterized by insulin resistance of glucose disposal and insulin hypersecretion, with preserved β-GS and rate sensitivity, i.e., the metabolic picture of obesity. In the T2D patients, insulin resistance was worse, and both β-GS and rate sensitivity were markedly impaired, thereby accounting for the hyperglycemia. Whereas the rate of appearance of oral glucose was similar in time course and total amount in all groups, in obese and T2D patients, EGP was higher at baseline and was incompletely suppressed during the meal, in some proportion to the degree of obesity, despite the hyperinsulinemia, thereby manifesting liver insulin resistance. In the T2D patients, the blunted rise in the prehepatic insulin-to-glucagon ratio during the first 2 h postmeal ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0321/-/DC1)) likely contributed to the elevated EGP.

One year after RYGB, at a time when drastic weight loss had occurred and body weight had stabilized, fasting plasma glucose was similar in ND and diabetic patients, but in the latter, 2-h glucose concentrations were significantly higher (5.93 ± 0.53 vs. 4.23 ± 0.31 mmol/L, *P* = 0.02), and mean glucose levels during the 5-h meal tended to be higher (Table 2), than in ND surgical patients. Insulin sensitivity improved in both surgical groups to levels close to those of the BMI-matched control group, i.e., in rough proportion to the weight loss and without reaching the values of the lean control subjects (Fig. 4). This result, obtained by tracer analysis of a dynamic test such as the MTT, confirms the findings of steady-state (clamp) measurements of insulin sensitivity (17), namely, that RYGB does not potentiate insulin action beyond, or independenty of, the effect on weight loss. β-GS worsened slightly in the ND surgical patients, whereas it doubled in the diabetic patients, in whom, however, it remained markedly inferior to that of BMI-matched ND subjects. This outcome prevailed despite the large increase in GLP-1 response, a consistent change after bariatric surgery (10–17), even as long as 10 years after surgery (33), which is quantitatively related to enhanced insulin secretion (19). In both groups, rate sensitivity improved postsurgery, likely reflecting the rapid plasma glucose excursions as well as, at least in part, the heightened GLP-1 surge ([Supplementary Fig. 2*A*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0321/-/DC1)). The latter finding is compatible with evidence that a rapid rather than delayed delivery of insulin improves glucose tolerance irrespective of the degree of insulin resistance (34), and may explain why meal tolerance in our patients was preserved 1 year after surgery despite the abnormal β-GS.

Thus, in T2D patients, recovery of both insulin sensitivity and glucose sensitivity was sizeable but incomplete, leaving behind a trace of glucose intolerance. These results confirm previous findings in both ND and T2D subjects, using a liquid formula meal and homeostasis model assessment of insulin resistance as a surrogate index of insulin resistance (35), or an oral glucose tolerance test (18). Further increments in both functions may occur if more weight is lost or as removal of the toxic effects of hyperglycemia continues; on the other hand, diabetes may relapse if weight is regained, if insulin resistance otherwise worsens, or if the disease itself should progress. In addition, as previously shown, in T2D patients, the outcome of glucose tolerance at 1 year post-RYGB may be better or worse than in the present series of patients depending on the initial degree of β-cell dysfunction (18).

As found by Bradley et al. (24) in ND subjects, RYGB drastically changed the shape of the glucose and hormonal responses to the meal, with a markedly biphasic pattern reminiscent of a dumping syndrome. Glucose fluxes confirmed that this was the consequence of the altered delivery of gastric contents to the peripheral circulation. A somewhat unexpected consequence of the altered pattern of transit of alimentary glucose was the reduced suppression of endogenous glucose release during the meal. Under euglycemic clamp conditions, hepatic and peripheral insulin resistance are typically somewhat interrelated and change consensually (36). A mixed meal, however, creates a hormonal makeover by stimulating both insulin and glucagon secretion, especially in diabetic patients in whom glucagon responses are exaggerated (37). In our surgical patients, both fasting insulin and fasting glucagon concentrations fell significantly postsurgery; correspondingly, fasting EGP did not change after the operation. After the meal, the prehepatic insulin-to-glucagon molar ratio normally rises in a time course roughly parallel to that of plasma glucose concentrations and ISRs; in T2D patients, the ratio shows the blunted initial rise followed by a sustained increase typical of the insulin secretory response ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0321/-/DC1)). After surgery, however, plasma glucagon rose sharply and remained above basal levels throughout the absorption period both in ND and T2D patients (Fig. 6), such that the prehepatic insulin-to-glucagon ratio, after peaking at ∼1 h postmeal, dropped rapidly to levels below presurgery ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0321/-/DC1)), in phase with the lower plasma glucose levels. Thus, the raised postmeal EGP was the integrated liver response to lower insulin, higher glucagon, and falling glucose levels. The correlation between the calculated index of hepatic insulin resistance and the glucagon response supports the role of the relative hyperglucagonemia ([Supplementary Fig. 2*B*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0321/-/DC1)). It should be noted that in the cited study in ND subjects (24), the time course of EGP was similar to ours but the glucagon response was rather flat and unchanged from presurgery. This difference may be due to the use of a smaller meal (∼300 kcal with only 9 g of protein compared with 500 kcal in the current study) and/or the shorter time interval from surgery (22 weeks), a time at which patients are typically still losing weight. The stimulus for the meal-induced hyperglucagonemia 1 year after surgery, which has been noted before (15), remains undetermined. Diminished paracrine control of α-cell activity by insulin (38) seems unlikely as the glucagon excursions in our patients were essentially synchronous with those of plasma insulin. Potential mechanisms are enhanced postprandial neural stimulation (39), overstimulation of glucagon release by GIP (40) or GLP-2 (41), and cosecretion of glucagon and GLP-1/GIP by intestinal cells (42). In our data, the postsurgery time courses of glucagon and GIP were in phase, lending some support to an effect of GIP on α-cells. It should also be considered that the standard glucagon assay has limitations in terms of its ability to discriminate between pancreatic glucagon and other sources (43).

Although gastric emptying was not measured in the present studies, our previous work in post-RYGB patients (using a scintigraphic technique) has confirmed that the operation causes accelerated gastric emptying even 14–26 months later (44); this result is fully compatible with the accelerated appearance of oral glucose in the present series.

In summary, the long-term outcome of RYGB is a comparable weight reduction and a proportional improvement in insulin sensitivity in ND and well-controlled, recent-onset diabetic patients. In both, delivery of oral glucose to the peripheral circulation is maintained in quantity but strikingly changed in time course, with a "dumping" pattern resulting from the modified anatomy. Glucose tolerance is preserved in ND and much improved in diabetic patients; in the latter, a detectable degree of glucose intolerance persists. In diabetic patients, β-cell function improves presumably as a combined result of reversal of glucose toxicity, lower insulin secretory burden, and incretin-mediated potentiation, but β-GS remains subnormal. This may predispose patients to recurrent diabetes. The surgery-induced change in glucose delivery triggers not only a heightened GLP-1 response but also an exaggerated glucagon response synchronous with the glucose excursions, which likely contributes to maintain euglycemia through elevated rates of EGP.

## ACKNOWLEDGMENTS

This work was aided in part by a grant from the Italian Ministry of Education, University and Research (PRIN 2010329EKE).

No potential conflicts of interest relevant to this article were reported.

S.C. designed the study, performed the in vivo studies, analyzed the data, and wrote the manuscript. E.M. performed the in vivo studies and contributed to the discussion. A.G. and D.C. analyzed the tracer data. J.J.H. was responsible for hormone measurements and contributed to the discussion. B.A. and M.N. performed the in vivo studies. S.B. was responsible for laboratory measurements. M.A. operated on the patients. A.M. analyzed the β-cell function data. E.F. contributed to the design of the study, analyzed the data, and wrote the manuscript. S.C. and E.F. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

The authors are grateful to Dr. Laura Manca (University of Pisa) for help with the statistical analysis.

# REFERENCES


abstract: A report from the Abcam genome stability conference 'Maintenance of Genome Stability', Jolly Beach Resort, Antigua, 8-11 March 2010.
author: Kyle M Miller
date: 2010
institute: 1The Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
title: Advances in understanding genome maintenance

'Maintenance of Genome Stability' is a biennial meeting that brings together, in a fantastic venue, diverse researchers working on how the integrity of genomes is maintained. Topics included DNA repair pathways, replication and recombination, and common themes included how these processes are regulated during the cell cycle, in the context of their chromatin or genomic location, and their involvement in cancer. Here, I summarize some highlights of the meeting, which included large-scale genomic studies, work on post-translational modifications in genome maintenance, and insights into new mechanisms and proteins involved in DNA repair pathways, telomeres and cancer.

# Mass spectrometry proteomics

Post-translational modifications in DNA damage signaling was a common theme throughout the meeting. Jiri Bartek (Centre for Genotoxic Stress Research, Copenhagen, Denmark) presented a genome-wide proteomics screen, using quantitative mass spectrometry (stable isotope labeling with amino acids in cell culture, SILAC), for phosphorylations of nuclear proteins that change following DNA damage in human cells. Over 7,000 phosphorylation sites were detected, with 2,000 being novel and not described in other proteomic screens. The power of this screen was that it analyzed the temporal regulation of phosphorylations after DNA damage. To accomplish this, cells were analyzed at time zero (no damage) and then at 5 minutes, 20 minutes, 1 hour and 8 hours after DNA damage. Taking into account only those phosphorylations detected at all time points, almost 600 phosphorylations were found to change by over twofold. Induced phosphorylations and dephosphorylations were detected. Phosphorylations that increased at early time points after DNA damage were enriched in substrates for the kinase ataxia-telangiectasia mutated (ATM). Looking at the amino acid sequences surrounding the phosphorylation sites, a new phosphorylation consensus sequence, SxxQ, was determined that does not seem to be mediated by any known kinase. This dataset is very likely to be a useful resource for researchers interested in phosphorylation in the DNA damage response (DDR).

Post-translational modifications other than phosphorylation are also important for the DDR, and it is clear that ubiquitylation has a key role in DNA damage signaling. Work from Jiri Lukas' group (Center for Genotoxic Stress Research, Copenhagen, Denmark) and others have added strong evidence for this notion by identifying two ubiquitin E3 ligases, RNF8 and RNF168, as important regulators of DNA damage signaling. To gain insights into the substrates of these two enzymes, Lukas' group performed quantitative mass spectrometry (SILAC) of 6 × His-Flag-Ub-containing complexes from cells lacking RNF8 or RNF168. Many DDR proteins were found to be ubiquitylated in an RNF8- or RNF168-dependent manner. Of the histones, only histone H2A and histone H2AX were purified and verified as substrates. Therefore, ubiquitin not only decorates the chromatin around a double-strand break (DSB) but also is conjugated to many signaling proteins, which again highlights the importance of this modification in the DDR. An understanding of how ubiquitylation affects DNA repair and signaling proteins to bring about proper repair is guaranteed to be a continued topic of research.

Chromatin is an important component in promoting genomic and epigenomic stability. Anja Groth (BRIC, University of Copenhagen, Denmark) discussed recent findings from her group showing the histone modifications on histones H3 and H4 that are associated with the histone chaperone Asf1b in S phase or in response to replication impaired by hydroxyurea (HU). Complexes of Asf1b containing histones H3 and H4 were purified and quantified by mass spectrometry. Groth described how all Asf1b-bound H4 molecules were acetylated on Lys5 and Lys12; histone H3 was 30% acetylated on Lys18 and 20% acetylated on Lys14; and H3 was also mono-methylated at Lys9. Acetylation of H3 on Lys56 was barely detectable, suggesting that this mark is not a general mark of histones in S phase. During replication stress (HU), methylation of H3 on Lys9 (H3K9me1) increased significantly in Asf1b complexes. Thus, this epigenetic mark is deregulated after replication stress, and Asf1b seems to have a role in sequestering H3 molecules containing this mark. As H3K9me1 can affect epigenetic silencing, this work suggests that replication stress could affect epigenomic integrity through Asf1b.

# Site-specific DNA double-strand breaks

The DNA damage field has long awaited the arrival of a robust system for studying site-specific DSBs in mammalian cells. This wait might be over because two research groups revealed such systems. Gaelle Legube (University of Toulouse, France) showed a new site-specific DSB system that uses a rare cutting restriction enzyme that is regulatable. The break sites are found throughout the genome and can be analyzed simultaneously. Chromatin immunoprecipitation microarray (ChIP-chip) analysis demonstrated robust phospho-histone H2AX (γH2AX) formation at many predicted genomic break sites. Legube found that large γH2AX domains formed bidirectionally from the DNA breaks but were not uniform or symmetrical, suggesting genomic boundaries that limit γH2AX spreading. Active transcription still occurred in γH2AX domains, although the level of γH2AX was reduced around transcription start sites, possibly because of increased histone dynamics.

Roger Greenberg (University of Pennsylvania, Philadelphia, USA) presented data from a system that uses an integrated Lac operator (LacO) array that is positioned approximately 4 kb upstream of a transcriptionally active site, which can be monitored in real time for DSB responses and nascent transcript formation. In this system, a LacI-FokI nuclease fusion protein is expressed that site-specifically binds the LacO array and creates site-specific damage. This is a great tool to look at the effects of DNA damage on transcription. Indeed, Greenberg's group found that, following DNA damage, transcription was rapidly halted, with a concomitant compaction of the genomic region due to DSB-induced inhibition of RNA polymerase II elongation. These effects were all dependent on the DDR kinase ATM. The transcriptional silencing occurred *in cis*, as creation of DNA damage outside this region did not affect transcription from the reporter locus. Silencing was rapidly reversible following DSB repair, as were all the effects described. Finally, ubiquitylated histone H2A was detected at this region and its deubiquitylation by the ubiquitin-specific peptidase 16 (USP16) was required for resumption of transcription following DNA repair. Thus, DNA damage within multiple kilobases of a transcriptionally active locus results in transcriptional repression that is dependent on ATM and ubiquitin.

# Novel proteins and processes in genome maintenance

## Deubiquitylation in the DNA damage response

As ubiquitin conjugation has been linked with DNA damage surveillance and repair, systems must be present to modulate or counteract these pathways. Daniel Durocher's group (Samuel Lunenfeld Research Institute, Toronto, Canada) has pioneered the use of genome-wide screens using small interfering RNA (siRNA) to look microscopically at how knockdown of individual genes affects DNA damage signaling and protein localization at DNA damage sites. Durocher's group previously identified two ubiquitin E3 ligase enzymes, RNF8 and RNF168, that are required for DNA repair through their combined ubiquitylation of substrates, including histones, at sites of DNA damage. Durocher's group has now performed a siRNA screen targeting the human deubiquitylating enzymes (DUBs) to analyze how these enzymes can affect ubiquitin conjugates at DNA damage sites. Daniel Durocher presented in a talk his groups findings from this screen that revealed several candidate genes whose knockdown resulted in the accumulation of ubiquitin conjugates at unrepaired DNA damage. Thus, these data show that systems do indeed exist that function to deubiquitylate substrates at sites of DNA damage. Defining the DUBs and their key targets that function in the DDR, as well as determining how ubiquitin affects these pathways, are important questions in the field. As the ubiquitin pathway is extremely complex, answers to these questions will undoubtedly uncover many surprises and interesting biology that will be important in advancing our understanding of the role of ubiquitin in genome maintenance.

## Fanconi anemia

Fanconi anemia (FA) is a rare genetic disorder resulting in an increased incidence of some cancers and heightened cellular sensitivity to inter-strand DNA cross-linking agents. John Rouse (University of Dundee, UK) and Josef Jiricny (University of Zurich, Switzerland) introduced a new FA-associated protein, REND1/KIAA1018, which functions as a 5' flap endonuclease and an exonuclease in the repair of inter-strand crosslinks. Following DNA damage, this protein forms foci by binding the ubiquitin-conjugated form of Fanconi anemia group D2 protein (FANCD2), revealing that it functions downstream of FANCD2. Loss of REND1/KIAA1018 resulted in hypersensitivity to inter-strand DNA crosslinks, showing that it is a *bona fide* participant of the FA pathway. Whether mutations in REND1/KIAA1018 are found in human patients suffering from Fanconi anemia is yet to be determined but this work suggests that there may be additional, as-yet-unidentified proteins that function in this complex repair pathway, which is essential for maintaining genome stability.

## Telomeres and cancer

Aneuploidy is prominent in many cancers and it has been hypothesized that tetraploidy initializes these events. Dysfunctional telomeres are also a common initiating event in cancer but a link between these two processes has not yet been established. Titia de Lange (Rockefeller University, New York, USA) presented data showing how telomere dysfunction can cause endoreduplication, resulting in tetraploidy in p53-deficient cells. Deprotection of telomeres by the absence of the telomere-associated Pot1a/b proteins resulted in cell cycle arrest, which was dependent on the protein ataxia-telangiectasia and Rad3-related (ATR), and tetraploidy. Using a powerful microscopy technique, fluorescence ubiquitination cell cycle indicator (FUCCI), de Lange showed *in vivo* data of these cells degrading geminin and re-expressing the origin licensing factor Cdt1 in the absence of mitosis, resulting in endoreduplication. When telomere end-protection was reintroduced, these tetraploid cells could resume growth. This mechanism of tetraploidization is not limited to telomere deprotection because DNA damage can result in the same outcome. Thus, these data reveal how telomeres and DNA damage can result in tetraploidy, a cellular state seen in cancer and implicated in tumorigenesis.

This successful conference series has become a must-attend meeting in the field of genome stability. Although details are unavailable for 2012, this meeting is sure to supply great science to people who are interested in the mechanisms that govern genome integrity.

## Acknowledgements

I thank the Wellcome Trust for providing my funding to attend this meeting.


abstract: Neutrophil extracellular chromatin traps (NETs) are a recently described mechanism of innate immune responses to bacteria and fungi. Evidence indicates that NETs are induced by inflammation, that they contribute to diverse disease pathologies, and that they associate with bactericidal substances. Genomic DNA is released in NETs, leading to a cell death that has been labeled NETosis. Although NETosis clearly differs from apoptosis, the classical form of cell death, recent experiments indicate a connection between NETosis and autophagy. The regulated deployment of NETs may require covalent modification of histones, the basic DNA-binding proteins that organize chromatin in the cell's nucleus and within NETs. Histone modification by peptidylarginine deiminase 4 (PAD4) is necessary for NET release. The functions of additional histone modifications, however, remain to be tested.
author: Indira Neeli; Marko Radic
date: 2012
institute: 1Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA
references:
title: Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps

See related research by Liu *et al.*, <http://arthritis-research.com/content/14/1/R25>

Less than a decade since their discovery, neutrophil extracellular traps (NETs) remain in the headlines. Initially, interest focused on the structure of extracellular NET chromatin and its capacity to capture and damage bacteria. Soon, however, researchers began to see the implications of extracellular chromatin for the development of autoimmune diseases. One quintessential autoimmune disease, systemic lupus erythematosus (SLE), is known to arise together with autoantibodies to DNA and chromatin, although the immediate trigger for the production of these autoantibodies is unclear. A connection between NETs and autoimmunity was made by discovering that histones, a set of proteins that act as a structural harness for DNA in chromatin, are modified by peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginines to citrullines. Researchers had long suspected that autoantigen modifications could provide the initial stimuli in autoimmunity because subtle alterations in a protein's primary sequence can break tolerance. PAD4 is implicated in the development of rheumatoid arthritis (RA) because the most reliable clinical test for RA uses the detection of anti-citrulline antibodies in the sera of patients.

In a sophisticated set of experiments reported in the previous issue of *Arthritis Research & Therapy*, Liu and colleagues \[1\] accomplished an extensive inventory of post-translational modifications in NET histones. The researchers induced NETs from human neutrophils, as well as two cell lines that assume neutrophil-like characteristics, and used a panel of 40 commercially available antisera to identify histone modifications that arise in parallel with NETs. Stimuli that were used to elicit NET release also induced histone H3 and H4 citrullination in human neutrophils and the EPRO cell line. However, other modifications such as histone H4 lysine 20 methylation and H4 lysine 16 acetylation showed inconsistent results in neutrophils versus the EPRO cells. To survey histone modifications, Liu and colleagues \[1\] confronted technical difficulties in that histone amino terminal tails contain the highest concentration of histone modifications yet are also highly susceptible to proteases secreted by activated neutrophils \[2,3\]. The histone tails act as flexible tethers that organize chromatin into higher-order structures. Interestingly, purified NETs failed to induce an immune response in mice, although a subset of SLE sera reacted strongly with citrullinated histone H3 \[1\]. Therefore, mechanisms that regulate histone modification deserve further attention.

Neeli and colleagues \[4\] were the first to identify citrullinated histone H3 in NETs, a discovery that was confirmed by others \[5\]. Neeli and colleagues \[4\] provided a second important insight, namely that PAD4-citrullinated histone H3 is a reliable marker of inflammation. Thus, it became clear that the release of NETs is not an 'accident' caused by a barrage of proteases and reactive oxygen species unleashed from neutrophils. Instead, production of NETs requires enzymatic activity and input from neutrophil surface receptors and the cytoskeleton \[6\]. By analyzing PAD4-deficient mice, Li and colleagues \[7\] demonstrated that PAD4 is essential for the production of NETs in response to bacterial infections. The regulation of PAD4 activity thus moved to the forefront of the research on NETs.

It is now clear that NET release takes advantage of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and the main granule proteases to trigger and construct the extended chromatin network \[3,8\]. In addition, myeloperoxidase is found in NETs after their release from the cells, and this enzyme and its products are the main components in NETs that kill bacteria \[9\]. In a notable study from the labs of Banchereau and Pascual \[10\], it was reported that SLE neutrophils are poised to undergo NETosis upon stimulation with anti-ribonucleo-protein autoantibodies and that NETs released by these neutrophils contain LL37 and HMGB-1, well-known stimulators of immune responses. In subsequent analyses using sera from patients with connective tissue disease, anti-citrullinated histone antibodies were observed in Felty's syndrome, a rare disorder that shares serologic features with RA and SLE, whereas such autoantibodies were infrequent in SLE and RA \[11\]. These findings indicate that the process of NETosis is highly relevant to the development of human autoimmune responses, although a direct cause and effect may not connect the release of NETs to the production of autoantibodies.

The detailed characterization of NET histone modifications, as accomplished by Liu and colleagues \[1\], invites speculations about the possible functions of these modifications. Several questions deserve further study: Will NET histone modifications, such as methylation, acetylation, and citrullination, be found to participate in gene regulation that sets the stage for NET release? Will the primary function of histone modifications turn out to be the decondensation of nuclear chromatin that is required for NETs expand to their optimal size and internal structure? Alternatively, NET histone modifications may serve non-traditional purposes. For example, certain modifications may anchor other NET components such as elastase, LL37, or myeloperoxidase to the chromatin meshwork. Unique modifications in NETs may attract phagocytes and stimulate them to ingest the trapped microorganisms. Other histone modifications may activate or dampen the inflammatory response by acting on innate pattern recognition receptors. The answers to these questions will, no doubt, keep research on NETs in leading immunology and microbiology journals for years to come.

# Abbreviations

NET: neutrophil extracellular trap; PAD4: peptidylarginine deiminase 4; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

Both authors contributed to the writing and editing of the text.

## Acknowledgements

The authors acknowledge the research support from the Lupus Research Institute of New York and the Dana Foundation for Human Immunology. We apologize for our highly focused and therefore incomplete survey of research accomplishments in NET research.


date: 2017
title: Marijuana associated with three-fold risk of death from hypertension

# introduction

Marijuana use is associated with a three-fold risk of death from hypertension, according to research published recently in the European Journal of Preventive Cardiology.

'Steps are being taken towards legalisation and decriminalisation of marijuana in the United States, and rates of recreational marijuana use may increase substantially as a result', said lead author Barbara A Yankey, a PhD student in the School of Public Health, Georgia State University, Atlanta, US. 'However, there is little research on the impact of marijuana use on cardiovascular and cerebrovascular mortality.'

In the absence of longitudinal data on marijuana use, the researchers designed a retrospective follow-up study of NHANES (National Health and Nutrition Examination Survey) involving participants aged 20 years and older. In 2005–2006, participants were asked if they had ever used marijuana. Those who answered 'yes' were considered marijuana users. Participants reported the age when they first tried marijuana and this was subtracted from their current age to calculate the duration of use.

Information on marijuana use was merged with mortality data in 2011 from the National Centre for Health Statistics. The researchers estimated the associations of marijuana use and duration of use with death from hypertension, heart disease and cerebrovascular disease, controlling for cigarette use and demographic variables including gender, age and ethnicity. Death from hypertension included multiple causes such as primary hypertension and hypertensive renal disease.

Among a total of 1 213 participants, 34% used neither marijuana nor cigarettes, 21% used only marijuana, 20% used marijuana and smoked cigarettes, 16% used marijuana and were past-smokers, 5% were past-smokers and 4% only smoked cigarettes. The average duration of marijuana use was 11.5 years.

Marijuana users had a higher risk of dying from hypertension. Compared to non-users, marijuana users had a 3.42-times higher risk of death from hypertension and a 1.04 greater risk for each year of use. There was no association between marijuana use and death from heart disease or cerebrovascular disease.

Ms Yankey pointed out that there were limitations to the way marijuana use was estimated. For example, it cannot be certain that participants used marijuana continuously since they first tried it.

She said: 'Our results suggest a possible risk of hypertension mortality from marijuana use. This is not surprising since marijuana is known to have a number of effects on the cardiovascular system. Marijuana stimulates the sympathetic nervous system, leading to increases in heart rate, blood pressure and oxygen demand. Emergency rooms have reported cases of angina and heart attacks after marijuana use.'

'We found higher estimated cardiovascular risks associated with marijuana use than cigarette smoking', said Ms Yankey. 'This indicates that marijuana use may carry even heavier consequences on the cardiovascular system than that already established for cigarette smoking. However, the number of smokers in our study was small and this needs to be examined in a larger study.'

'Needless to say, the detrimental effects of marijuana on brain function far exceed that of cigarette smoking', she added. Ms Yankey said it was crucial to understand the effects of marijuana on health so that policy makers and individuals could make informed decisions.

She said: 'Support for liberal marijuana use is partly due to claims that it is beneficial and possibly not harmful to health. With the impending increase in recreational marijuana use it is important to establish whether any health benefits outweigh the potential health, social and economic risks. If marijuana use is implicated in cardiovascular diseases and deaths, then it rests on the health community and policy makers to protect the public.'

Source: European Society of Cardiology Press Office


abstract: # Introduction
  .
  The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of *Klotho*, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.
  .
  # Methods
  .
  Rat nucleus pulposus cells were cultured under normoxic (21% O~2~) or hypoxic (2% O~2~) conditions, and the expression and promoter activity of Wnt signaling and *Klotho* were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated β-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.
  .
  # Results
  .
  Nucleus pulposus cells exhibited increased β-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed *in vivo*, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells.
  .
  # Conclusions
  .
  These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.
author: Akihiko Hiyama; Fumiyuki Arai; Daisuke Sakai; Katsuya Yokoyama; Joji Mochida
date: 2012
institute: 1Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan; 2Research Center for Regenerative Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
references:
title: The effects of oxygen tension and antiaging factor *Klotho* on Wnt signaling in nucleus pulposus cells

# Introduction

Regenerative therapy for intervertebral disc degeneration has been recently reported \[1-3\]. Cell-based therapies for tissue regeneration offer an attractive alternative to current conservative, surgical, pharmaceutical, or gene-therapy interventions. However, to clarify the mechanism underlying low-back pain, the molecular mechanisms involved in intervertebral disc degeneration must be identified.

Wnt/β-catenin (hereafter called Wnt) signaling is thought to be involved in the maintenance and destruction of bone and cartilage. Dysregulation of members of this signaling family has been described in osteoarthritis \[4-6\]. Wnts are secreted glycoproteins crucial for the development and homeostatic renewal of many tissues and for chondrocyte and osteoblast development. In the presence of Wnt ligands, Wnts activate a number of different signaling pathways via distinct receptors and downstream effectors that mediate effects on gene transcription \[7-9\]. Although Wnt signals regulate the balance between catabolic factors and anabolic factors in intervertebral discs \[10,11\], their regulation (upstream or downstream) in nucleus pulposus cells and the corresponding signaling mechanisms are unknown.

One of the primary causes of intervertebral disc degeneration is thought to be failure of the nutrient supply to intervertebral disc cells because of structural changes to the cartilage endplate \[12\]. The O~2~ levels in the nucleus pulposus may be 1% to 5%, and disc-cell metabolism can vary with O~2~ concentration. The activity of disc cells is very sensitive to changes in extracellular oxygen and pH. However, a little information is known about the effect of oxygen tension on nucleus pulposus cells \[13\]. Accordingly, more data are needed to determine whether a low oxygen tension is beneficial or detrimental in the culture of nucleus pulposus cells.

The jelly-like nucleus pulposus (notochord) in the middle of the disc is composed of proteoglycan and functions to disperse the normal loading forces experienced by the spine, acting as a shock absorber to maintain the trunk. However, changes in proteoglycan concentration during age-related disc degeneration are of critical importance. During embryogenesis of the intervertebral disc cells, the cells of the notochord play a critical role in initiating tissue formation and may be directly responsible for development of the nucleus pulposus. In some species, including humans, notochordal cells may eventually be lost and are replaced by chondrocyte-like cells \[14,15\]. By the age of 60 to 80 years, the intervertebral disc is composed entirely of fiber \[16,17\]. Accordingly, aging is another risk factor for intervertebral disc degeneration. During degenerative disc disease, loss of disc cells, limited proteoglycan synthesis, and a shift toward synthesis of a fibrotic matrix occur. *Klotho*, a newly identified antiaging gene, has attracted recent attention. The *Klotho* gene encodes a single-pass transmembrane protein. *Klotho* is predominantly expressed in the kidney, but it is also expressed in the brain, parathyroid gland, and heart of normal subjects \[18-20\]. The *Klotho* gene plays a critical role in regulating aging and in the development of age-related diseases in mammals. Loss of *Klotho* can result in multiple aging-like phenotypes \[21,22\], and conversely, the overexpression of *Klotho* in *Klotho*^-/-^ mice extends their life span \[23\]. *Klotho* gene polymorphisms in humans are associated with pathophysiologic bone loss with aging \[24\], spondylosis \[25\], osteocalcin levels \[26\], and bone mineral density \[27\]. However, no reports exist of the expression of Klotho protein in the intervertebral disc. Moreover, factors responsible for age-associated impairment of intervertebral disc are poorly understood.

In the previous study, we focused on assessing the relation among oxygen tension, klotho, and Wnt signaling and attempted to examine the biologic mechanisms (upstream or downstream) of Wnt signaling in nucleus pulposus cells. The Wnt-signal downstream promoter may be induced by oxygen tension or an age-related gene. Therefore, the purpose of the present investigation was threefold: to determine how Wnt signaling in nucleus pulposus cells is regulated by oxygen tension; to determine the effects of the aging-suppressor gene *Klotho* on Wnt signaling; and to investigate the relation between disc degeneration (maintenance of cell proliferation and matrix synthesis) and *Klotho* expression. These findings provide new insight into the regulation and maintenance of cell proliferation and matrix synthesis by *Klotho* and Wnt signaling cross-talk in nucleus pulposus cells.

# Materials and methods

All animal experiments were performed with approval from Tokai University animal study institutional review board.

## Reagents and plasmids

Plasmids were kindly provided by Dr. Raymond Poon (Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada) (plasmid encoding wild-type (WT) β-catenin and the backbone), Ilsa Rovira (Cardiology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA) (plasmid encoding Flag-tagged full-length Klotho) \[28\], Dr. Young Han Lee (Institute of Biomedical Science and Technology, Konkuk University Hospital, Seoul, South Korea) (plasmids encoding luciferase-tagged Klotho constructs, Del-1, Del-2, Del-3, and Del-4) \[29\], Dr. Michael C. Naski (University of Texas Health Science Center at San Antonio, San Antonio, TX, USA) (plasmid encoding luciferase-tagged aggrecan) \[30\], Dr. Sean A. McCarthy (University of Michigan, Ann Arbor, MI, USA) (plasmid encoding Dickkopf-1 (Dkk1), Dkk2, Dkk3, and Dkk4 expression vectors or backbone vector), and Dr. Yoshihiko Yamada (Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Bethesda, MD, USA) (plasmid encoding luciferase-tagged type-II collagen) \[31\]. The constitutively active (CA)-GSK3β (no. 14754) and dominant-negative (DN) GSK3β (no. 14755) were purchased from Addgene (Cambridge, MA, USA). Topflash (optimal Tcf-binding site) and Fopflash (a promoter with a mutated Tcf-binding site) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). β-Catenin small interfering RNA (siRNA) (no. sc-29209) and control siRNA duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In the Klotho constructs, the TCF/LEF binding motifs within the klotho promoter were analyzed by using the Web-based tool for predicting transcription factor-binding sites in DNA sequences, Transcription Element Search System (TESS). As an internal transfection control, we used the empty vector pGL4.74 (Promega, Fitchburg, WI, USA) containing the *Renilla reniformis* luciferase genes. The amount of transfected plasmid, the pretransfection period after seeding, and the posttransfection period before harvesting were optimized for rat nucleus pulposus cells by using the pSVβ-galactosidase plasmid (Promega). We used 6-bromoindirubin-3'-oxime (BIO) (no. 361550; Calbiochem, San Diego, CA, USA) to examine the activity of the Wnt signaling activity. BIO is a cell-permeable, highly potent, selective, reversible, and ATP-competitive specific inhibitor of GSK-3α/β activity \[32\]. Recombinant Klotho protein (α-Klotho) (no. 100-53) was purchased from Peprotech (Rocky Hill, NJ, USA).

## Isolation of intervertebral disc cells

In total, 64 (32 female and 32 male) 12-week-old Sprague-Dawley rats were used for this study. None of the variables differed between females and males. Nucleus pulposus cells were isolated by using methods reported by Hiyama *et al.*, 2007 \[1\]. In brief, the rats were euthanized by injection of an excess of pentobarbital sodium (100 mg/kg) (Nembutal; Abbott Laboratories, Abbott Park, IL, USA). The spinal column was removed under aseptic conditions, and the lumbar intervertebral discs were separated under microscopy. The gel-like nucleus pulposus was separated from the annulus fibrosus. The obtained nucleus pulposus tissue was digested in a mixture of 0.01% trypsin and allowed to digest at 37°C for 15 minutes. The digested tissue was passed through a cell strainer (BD Biosciences, San Jose, CA, USA) with a pore size of 100 μm and was washed twice with phosphate-buffered saline (PBS; Gibco, Invitrogen, Carlsbad, CA, USA). The isolated cells were maintained in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen) and 10% fetal bovine serum (FBS; Gibco, Invitrogen) supplemented with antibiotics (1% penicillin/streptomycin) at 37°C in a humidified atmosphere of 5% CO~2~. When confluent, the nucleus pulposus cells were harvested and subcultured in 10-cm dishes. Cells were then counted and plated at the appropriate density. Because cells obtained from the rat intervertebral disc tissues were variable in morphology until passage 2 to 3, we used low-passage (\< 3) cells cultured in monolayers for all experiments.

## Cell culture in hypoxia

To induce hypoxia, nucleus pulposus cells were cultured in a mixture of 2% O~2~, 5% CO~2~, and 93% N~2~ for 24 hours. The plating density chosen was dependent on the requirements of the individual assays. The concentration of oxygen and the duration of the incubation period chosen for this study were based on the *in vitro* studies of Risbud *et al.* \[33\] and on information generated on the oxemic status of the disc *in vivo*.

## Immunofluorescence staining

Nucleus pulposus cells were plated in flat-bottom 96-well plates (5 × 10^3^ cells/well) and maintained in conditions of normoxia or hypoxia for 24 hours. After this treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (vol/vol) in PBS for 10 minutes, blocked with PBS containing 5% FBS, and incubated overnight at 4°C with antibodies against β-catenin (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA). After washing, the cells were incubated with anti-rabbit IgG Alexa Fluor 594 (red) and/or anti-goat Alexa Fluor 488 secondary (green) antibodies (Invitrogen) at a dilution of 1:50 and 10 μM 4',6-diamidino-2-phenylindole (DAPI) for 1 hour at room temperature for nuclear staining. The samples were observed with a fluorescence microscope connected to a digital imaging system. Similar experiments were performed to assess the expression of anti-rabbit-Klotho (1:200 dilution; Abcam, Cambridge, UK) and anti-goat-Klotho (1:50 dilution; Santa Cruz Biotechnology). Negative controls without the primary antibody were prepared.

## Immunohistologic studies

Freshly isolated rat spines were immediately fixed in 4% paraformaldehyde in PBS, decalcified, embedded in paraffin, sectioned, and assessed by histology and then immunostaining. Sagittal sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin. To localize Klotho, sections were incubated with anti-Klotho antibody (ab75023; Abcam) in 2% bovine serum albumin (BSA) in PBS at a dilution of 1:200 at 4°C overnight. After thoroughly washing the sections, the bound primary antibody was detected with a biotinylated universal secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:20 for 10 minutes at room temperature. Sections were incubated with a streptavidin/peroxidase complex for 5 minutes and washed with PBS, and the color was developed by using 3'-3-diaminobenzidine (DAB) (VECTASTAIN Universal Quick Kit; Vector Laboratories) and peroxidase DAB substrate kit (Vector), according to the manufacturer's protocol. Negative controls without the primary antibody (anti-Klotho) were prepared.

## 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

To measure disc-cell proliferation, a modified MTT assay was carried out as previously described \[26\]. In brief, exponentially growing nucleus pulposus cells were seeded in 24-well plates at 1.5 × 10^4^ cells/well. After treatment with Klotho (100 ng/ml), MTT diluted in serum-free DMEM was added to the culture medium to a final concentration of 0.5 mg/ml. No data showed the physiological protein concentration of Klotho in the nucleus pulposus cells. We therefore chose to use the concentration of Klotho protein (100 ng/ml) referred to in a previous study \[34\]. At the end of the incubation period (2 hours at 37°C), the medium was removed, and the precipitated formazan crystals were solubilized in dimethyl sulfoxide (DMSO). Product formation was measured by reading the absorbance at 590 nm by using a microplate reader (Pharmacia).

## Staining for senescence-associated β-galactosidase (β-gal)

To stain nucleus pulposus cells for senescence-associated β-gal activity, we used a cell-senescence histochemical staining kit (CS0030; Sigma-Aldrich, St. Louis, MO, USA) according to the previously described protocol \[26\]. Cells were treated for 24 hours with or without 100 ng/ml Klotho. To quantify the results, a minimum of 100 cells spanning five different microscopy fields were scored for staining, and results represent the mean ± standard deviation (SD).

## Cell-cycle analysis by flow cytometry

Nucleus pulposus cells were grown in 24-well plates under a humidified 5% CO~2~ atmosphere at 37°C. The seeding density was 5 × 10^4^ cells/ml. The cells were allowed to adhere for 24 hours in medium containing 2% FBS. The culture medium in each flask was then replaced with medium containing 0.5% FBS. Klotho (100 ng/ml) was added to this medium as a concentrated stock solution dissolved in DMSO. After an additional incubation period of 24 or 48 hours, the cell-cycle distribution of the nucleus pulposus cells was analyzed with flow cytometry after DNA staining with propidium iodide by using the CycleTEST PLUS kit (BD Pharmingen, San Diego, CA, USA). CELLQuest (BD Pharmingen) and ModFit LT (BD Pharmingen) software packages were used for cell acquisition and analysis. Each plot represents the analysis of 10,000 events. The histograms present typical results, and the percentages of cells in G~1~, S, and G~2~/M cell-cycle phases are shown as the means of triplicate measurements. The results from three individual experiments are shown.

## Total RNA extraction and real-time reverse transcriptase (RT)-PCR analysis

Total RNA was extracted from rat nucleus pulposus and annulus fibrosus cells or tissues by using the TRIzol RNA isolation protocol (Invitrogen). Before elution from the column, RNA was treated with RNase-free DNAse I. Total RNA (100 ng) was used as a template for the real-time PCR analyses. The cDNA was synthesized by the reverse transcription of mRNA by using the protocol described previously (26). The real-time PCR analyses were performed in triplicate by using 96-well plates with the Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). Two microliters of cDNA per sample was used as the template for real-time PCR: 1 μl of forward primer and 1 μl of reverse primer were added to 20 μl SYBR Green Master Mix. PCR reactions were performed in an Applied Biosystems 7500 Fast system, according to the manufacturer's instructions. All primers for *β-catenin*, *Klotho*, and *aggrecan* were synthesized by Takara Bio, Inc. (Tokyo, Japan): *β-catenin* (NCBI number: AF_121265.1): forward, 5'-GCCAGTGGATTCCGTACTGT-3' and reverse, 5'-GAGCTTGCTTTCCTGATTGC-3'; *Klotho* (NCBI number: NM_031336.1): forward, 5'-CGATGTTCGTGACAGCCAATG-3' and reverse, 5'-GTTGATGCCGTCCAACACGTAG-3'; and *aggrecan* (NCBI number: NM_022190.1): forward, 5'-TCCGCTGGTCTGATGGACAC-3' and reverse, 5'-CCAGATCATCACTACGCAGTCCTC-3'. To normalize each sample, a control gene (GAPDH) was used, and the arbitrary intensity threshold (C~t~) of amplification was computed. The expression scores were obtained by the ΔΔC~t~ calculation method.

## Gene-suppression studies by small interfering RNA (siRNA)

We silenced β-catenin expression in nucleus pulposus cells by using small interfering RNA (siRNA) technology. In brief, nucleus pulposus cells were transferred to 24-well plates at a density of 6 × 10^4^ cells/well 1 day before transfection. The next day, cells were treated with β-catenin siRNA or control siRNA duplexes at a final concentration of 100 to 500 ng by using Lipofectamine 2000. Cells also received Klotho promoter constructs and the pGL4.74 plasmid at the time of transfection. Six hours after transfection, the medium was replaced with complete growth medium, and the cells were allowed to recover for 18 hours. Cells were then cultured for 24 hours, and luciferase activity was measured.

## Methylation analysis

Genomic DNA was extracted from cells not treated with BIO and BIO-treated cells by using a FastPure DNA Kit (Takara Bio), and bisulfite treatment of genomic DNA was performed by using MethylEasy Xceed (Human Genetic Signatures) and following the manufacturers' instructions. The bisulfite-treated DNA was amplified by PCR with Takara *Taq* Hot Start Version (Takara Bio) and T-Vector pMD20 (Takara Bio) for cytosine-phosphate-guanosine (CpG)-rich regions around the rat *Klotho* gene. PCR products amplified by using Takara LA Taq HS were subcloned into T-Vector pMD20, and sequence analysis was performed. All methylation studies were performed by bisulfite modification of DNA, which converts all unmethylated CpG sites to uracil-phosphate-guanosine, leaving methylated CpGs intact. The DNA was then amplified with the following gene-specific primers: *Klotho*, forward, 5'-TGATGTGGGGATATTTTAGGA-3', and reverse, 5'-CAACAAATACAACRACAACAAA-3'. After sequencing of the amplified DNA, methylated CpGs were identified with visual inspection of the sequencing traces in the electropherograms and by comparison with sequencing traces of BIO-treated cell gene sequences relative to the reference in the control cell sequence.

## Western blotting analysis

After treatment, nucleus pulposus cells were immediately washed 3 times with ice-cold PBS and solubilized in lysis buffer. Proteins were prepared by using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). All wash buffers and the final resuspension buffer included × 1 protease inhibitor cocktail (Roche, Switzerland), NaF (5 m*M*), and Na~3~VO~4~ (200 μ*M*). Total lysates (30 to 60 μg of protein/lane) were subjected to electrophoresis by using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). The resolved proteins were transferred electrophoretically to nitrocellulose membrane "blots". The blots were blocked with 5% BSA in TBST (50 m*M* Tris, pH 7.6, 150 m*M* NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 5% BSA in TBST with anti-β-catenin (1:1,000; Cell Signaling) or anti-Klotho (1:1,000; Abcam). Immunolabeling was detected with electrochemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA). Quantification of Western blots was performed by using Image J pixel analysis (NIH Image software). Data from Western blots are presented as band density normalized to the loading control (actin).

## Transfections and dual luciferase assay

Nucleus pulposus cells were transferred to 24-well plates at a density of 6 × 10^4^ cells/well 1 day before transfection. In several experiments, cells were cotransfected with 100 to 500 ng of expression plasmids or the backbone vector, along with reporter plasmids. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent. After 24 hours, cells were maintained in either the hypoxic or the normoxic condition. Twenty-four hours after treatment, the cells were harvested, and a Dual-Luciferase reporter assay system (Promega) was used for the sequential measurements of firefly and *Renilla* luciferase activities. The results were normalized for transfection efficiency and are expressed as a relative ratio of luciferase to pGL4.74 activities (denoted as relative activity). To check the transfection efficiency, nucleus pulposus cells were transfected with plasmid encoding green fluorescent protein; the transfection efficiency for nucleus pulposus cells was 60% to 70%. In addition, the overexpression or silencing efficiency of the targets was confirmed at the mRNA level or protein level. The luciferase activities and relative ratios were quantified by using a Turner Designs Luminometer Model TD-20/20 instrument (Promega).

## Statistical analysis

Typically, data were compiled from at least three independent triplicate experiments, each performed on separate cultures and on separate occasions. We calculated and have displayed the responses as fold change (over the untreated control). Data are presented as mean ± SD. Comparisons of data between groups were performed by using the Student *t* test or ANOVA for assessing variance. Statistical significance (*P* \< 0.05) is denoted with an asterisk.

# Results

## Oxygen tension directly regulates Wnt signaling in nucleus pulposus cells

We first examined whether oxygen tension activates the transcriptional activity of the Wnt signal pathway by using the TCF- and β-catenin-dependent Topflash reporter plasmid in nucleus pulposus cells. Figure <a href="#F1" data-ref-type="fig">1A</a> and <a href="#F1" data-ref-type="fig">1B</a> shows that under the hypoxic condition, Topflash activity increased significantly by 1.3- to 1.5-fold in nucleus pulposus cells after 24 hours of incubation (*P* \< 0.05). Nucleus pulposus cells cotransfected with the WT-β-catenin expression plasmid, or treated with a different concentration of BIO, showed a dose-dependent increase in the activity of Topflash. Fopflash promoter (a promoter with a mutated TCF-binding site) activity was unresponsive to oxygen tension (data not shown). We next measured the relative expression level of β-catenin mRNA under normoxic and hypoxic conditions. We analyzed mRNA expression with real-time RT-PCR. The β-catenin mRNA level was threefold higher in nucleus pulposus cells cultured under the hypoxic (2% O~2~) compared with the normoxic condition (21% O~2~) (*P* \< 0.05) (Figure <a href="#F1" data-ref-type="fig">1C</a>). To confirm the dependence of β-catenin expression on oxygen tension, as shown in Figure <a href="#F1" data-ref-type="fig">1D</a>, we showed that total β-catenin level and the nuclear translocation of β-catenin increased to a greater extent in nucleus pulposus cells cultured under the hypoxic condition than in cells cultured under the normoxic condition. In the hypoxic environment, a significant increase in the levels of β-catenin protein was seen (Figure <a href="#F1" data-ref-type="fig">1D</a>; arrows). To explore the premise that oxygen tension regulates β-catenin protein expression, nucleus pulposus cells were treated with BIO under normoxic and hypoxic conditions. The expression of β-catenin protein in nucleus pulposus cells was induced by hypoxia (Figure <a href="#F1" data-ref-type="fig">1E</a>).

## Expression of Klotho by intervertebral disc

To examine whether Klotho is involved in intervertebral disc differentiation, we first determined the expression profiles of Klotho in intervertebral discs. Expression of the Klotho protein and mRNA was analyzed by immunostaining and real-time PCR. We immunostained discs in sagittal sections from 3-week-old (Figure <a href="#F2" data-ref-type="fig">2A, a-d, i</a>) and 12-week-old (Figure <a href="#F2" data-ref-type="fig">2A, e-h, j</a>) rats by using an antibody to Klotho. Klotho was expressed in cells of the nucleus pulposus (Figure <a href="#F2" data-ref-type="fig">2A, d</a>), but not of the annulus fibrosus (Figure <a href="#F2" data-ref-type="fig">2A, c</a>), in the 3-week-old rat discs. Klotho expression was higher in the annulus fibrosus of discs in 12-week-old rats compared with 3-week-old rats. The nucleus pulposus of 3-week-old rats (Figure <a href="#F2" data-ref-type="fig">2A, i</a>) was full of notochordal cells with large vacuoles, and much of the staining was nuclear, which contrasted with the appearance in samples from 12-week-old rats (Figure <a href="#F2" data-ref-type="fig">2A, j</a>). To determine the expression of Klotho in intervertebral discs, we also measured Klotho mRNA expression both in tissue or cells and found that it was significantly higher in nucleus pulposus than in annulus fibrosus (Figure <a href="#F2" data-ref-type="fig">2B</a>). We sought to determine whether Klotho and the Wnt signal could form a direct molecular complex in nucleus pulposus cells. We demonstrated that the subcellular distribution of Klotho and β-catenin proteins within BIO-treated cells overlapped (Figure <a href="#F2" data-ref-type="fig">2C</a>).

## Decreased Klotho transcriptional activity in nucleus pulposus cells treated with hypoxia

We first investigated the regulation of Klotho expression to determine whether a relation existed between Klotho and the oxemic status of the nucleus pulposus cell by analyzing the 1.83-kb promoter sequence of human Klotho and measuring the activities of different sizes of promoter constructs (Figure <a href="#F3" data-ref-type="fig">3</a>). Sequence analysis shows that the Klotho promoter contains 12 TCF/LEF-binding motifs (CTTTT, CTTTG, or CAAAG); that is, 12 TCF/LEF-binding motifs in the p-Klotho Del1 (-1830/+7) construct, but no TCF/LEF-binding motifs in the p-Klotho Del4 (-90/+7) construct. To analyze the promoter function further, we used luciferase reporter constructs containing -1830/+7 bp (p-Klotho-Del1), -930/+7 bp (p-Klotho-Del2), -415/+7 bp (p-Klotho-Del3), or -90/+7 bp (p-Klotho-Del4) constructs of the Klotho promoter \[29\]. We measured the basal activity of all four constructs in nucleus pulposus cells. Figure <a href="#F4" data-ref-type="fig">4A</a> shows that the p-Klotho-Del3 construct had maximal basal activity, and the p-Klotho-Del1 construct exhibited the lowest activity. We next examined the effect of oxygen tension on all Klotho promoter activities. Experiments using serial deletion constructs of the Klotho promoter under the hypoxic condition showed a significant decrease in the activity of the p-Klotho-Del1 and p-Klotho-Del2 promoters but not of the p-Klotho-Del3 and p-Klotho-Del4 promoters (Del1: normoxic 1.00 ± 0.15, hypoxic 0.71 ± 0.10; Del2: normoxic 1.71 ± 0.10, hypoxic 1.53 ± 0.01; Del3: normoxic 2.78 ± 0.02, hypoxic 2.62 ± 0.14; Del4: normoxic 2.59 ± 0.05, hypoxic 2.71 ± 0.12) (Figure <a href="#F4" data-ref-type="fig">4A</a>). To evaluate these results further, a p-Klotho-Del1 reporter plasmid (Klotho-luc) was transfected along with the pGL4.74 vector into nucleus pulposus cells, and the cells were stimulated with BIO under the normoxic and hypoxic conditions. Figure <a href="#F4" data-ref-type="fig">4B</a> shows that the Klotho promoter activity was increased by BIO treatment. Cells cultured under the hypoxic condition showed lower Klotho promoter activity compared with the normoxic condition. Real-time RT-PCR analysis also showed that hypoxia decreased the expression of Klotho mRNA to 0.5 to 0.7 to that in normoxia (Figure <a href="#F4" data-ref-type="fig">4C</a>). To determine whether a concomitant elevation in Klotho protein expression was associated with Wnt signaling, the cells were evaluated with immunofluorescence analysis (Figure <a href="#F4" data-ref-type="fig">4D</a>) and Western blotting (Figure <a href="#F4" data-ref-type="fig">4E</a>). As shown in Figure <a href="#F4" data-ref-type="fig">4D</a>, immunofluorescence analysis with an anti-Klotho antibody showed that BIO treatment (1.0 μ*M*, 24 hours) promoted the nuclear translocation of Klotho more strongly in nucleus pulposus cells than in untreated cells. Klotho was localized to the nucleus in cells treated with BIO (arrows). Similarly, immunoblotting analysis of nucleus pulposus cells treated with BIO (1.0 μ*M*, 24 hours) showed more Klotho protein than in control nucleus pulposus cells (Figure <a href="#F4" data-ref-type="fig">4D</a>).

## Regulation of Klotho by Wnt signaling in nucleus pulposus cells

Although Wnt signaling has also been shown to have a role in nucleus pulposus cells, interactions between Wnt signaling and Klotho in nucleus pulposus cells have not been previously described. Initial experiments were performed to investigate the role of Wnt signaling in the transcriptional activity of Klotho in nucleus pulposus cells. Nucleus pulposus cells were transiently transfected with plasmids encoding Klotho along with WT-β-catenin expression plasmid. Figure <a href="#F5" data-ref-type="fig">5A</a> shows that forced expression of β-catenin under both normoxic and hypoxic conditions significantly induced Klotho promoter activity (*P* \< 0.05). To validate these findings, we performed loss-of-function experiments by using siRNA for β-catenin. Suppression of gene expression was confirmed by real-time RT-PCR (data not shown). Under the normoxic condition, Klotho promoter activity was inhibited in nucleus pulposus cells cotransfected with β-catenin siRNA (*P* \< 0.05; 100 ng, *P* = 0.005; 300 ng, *P* = 0.002; 500 ng, *P* = 0.0001) (Figure <a href="#F5" data-ref-type="fig">5B</a>). We also examined the effect of GSK3β treatment on Klotho promoter activity. Nucleus pulposus cells were transiently cotransfected with plasmids encoding CA-GSK3β (Figure <a href="#F5" data-ref-type="fig">5C</a>) or DN-GSK3β (Figure <a href="#F5" data-ref-type="fig">5D</a>). Klotho promoter activity was decreased in a dose-dependent manner by the CA-GSK3β expression plasmid (*P* \< 0.05) but not by the DN-GSK3β expression plasmid. These results suggest that Klotho is downstream of β-catenin signaling in nucleus pulposus cells. To confirm whether Dkk, a secretory protein that inhibits Wnt signaling by disrupting the interactions between Wnt and Fz, regulates the transcriptional activity of Klotho, we assessed the transcriptional activity of Klotho after treatment with Dkk1, 2, 3, or 4 (500 ng) expression plasmids or backbone vector for 24 hours. As shown in Additional file <a href="#S1" data-ref-type="supplementary-material">1</a> Klotho activity was not affected by Dkk treatment.

## Klotho suppression of the proliferation of nucleus pulposus cells

Proliferation of nucleus pulposus cells was decreased significantly by Klotho at 48 hours but not 24 hours after the treatment (*P* \< 0.05 and *P* = 0.24, respectively) (Figure <a href="#F6" data-ref-type="fig">6A</a>). To test for cell senescence, we stained cells for the expression of the senescence-associated protein, β-gal. As shown in Figure <a href="#F6" data-ref-type="fig">6B</a>, Klotho treatment for 24 hours did not change the percentage of senescence-associated β-gal-positive nucleus pulposus cells (arrow in control, 32.8% ± 6.4%; Klotho, 35.4% ± 8.8%). In addition, Klotho exposure did not increase the level of apoptosis (data not shown). To analyze the methylation status of the CpG islands in the Klotho promoter region, bisulfite genomic sequencing (BGS) analysis was performed in BIO-treated nucleus pulposus cells. As shown in Figure <a href="#F6" data-ref-type="fig">6C</a>, a dense GC-rich region was identified around the -200 bp site of Klotho. To determine the methylation density of the Klotho promoter region, which encompasses 61 CpGs, we performed BGS analysis of BIO-treated and untreated nucleus pulposus cells. The methylation of the Klotho promoter region was much less extensive in the BIO-treated cells with activated Wnt signaling (that is, activation of Wnt signaling tended to be associated with unmethylated CpG islands). These data strongly suggest that the transcriptional activation of Klotho is associated with aberrant promoter demethylation by Wnt signaling in nucleus pulposus cells.

With flow cytometry, we evaluated whether the cell-cycle status was affected by Klotho (100 ng) treatment at different times after treatment. The number of cells in the G~1~ phase was significantly decreased in the presence of Klotho compared with untreated cells (24 hours, *P* = 0.035; 48 hours, *P* \< 0.05). These results showed that activation of Klotho can suppress the proliferation of nucleus pulposus cells and may induce cells to enter a resting phase, the G~0~ phase (Figure <a href="#F6" data-ref-type="fig">6D</a>).

## Functional role of Klotho and Wnt signaling crosstalk in nucleus pulposus

To investigate the possible mechanisms underlying the actions of Klotho in the induction of nucleus pulposus cell markers, we next examined the effect of Klotho treatment on expression of aggrecan (Figure <a href="#F7" data-ref-type="fig">7A</a>) and collagen type II (Figure <a href="#F7" data-ref-type="fig">7B</a>) under the normoxic condition. Cotransfection of nucleus pulposus cells with a Klotho expression plasmid significantly decreased aggrecan reporter activity (Klotho 100 ng, *P* = 0.085; Klotho 300 ng, *P* = 0.011; Klotho 500 ng, *P* = 0.001) but not collagen type-II reporter activity (Klotho 100 ng, *P* = 0.723; Klotho 300 ng, *P* = 0.308; Klotho 500 ng, *P* = 0.126). To confirm the regulatory role of Klotho in the expression of aggrecan and collagen type II, we performed real-time PCR to examine changes in the expression of aggrecan and collagen type II in nucleus pulposus cells. The results showed that treatment with 500 ng of Klotho for 24 hours also decreased aggrecan mRNA levels in nucleus pulposus cells, as shown by real-time PCR (*P* \< 0.05; *P* = 0.004) (data not shown for collagen type II) (Figure <a href="#F7" data-ref-type="fig">7C</a>). In addition, we used immunofluorescence analysis and Western blotting to confirm the role of Klotho in aggrecan protein expression. The results demonstrated that Klotho treatment (100 ng/ml, 24 hours) decreased aggrecan protein levels to a greater extent in nucleus pulposus cells than in control cells (data not shown). Finally, to confirm the relation between Wnt signaling and Klotho and to assess whether Klotho inhibits Wnt signaling in nucleus pulposus cells, we used a Klotho expression plasmid in the Topflash reporter assay. Topflash reporter activity was decreased by Klotho treatment in nucleus pulposus cells (normoxic: Klotho 100 ng, *P* = 0.089; Klotho 300 ng, *P* = 0.044; Klotho 500 ng, *P* = 0.0003; hypoxic: Klotho 100 ng, *P* = 0.001; Klotho 300 ng, *P* = 0.002; Klotho 500 ng, *P* = 0.0001) (Figure <a href="#F7" data-ref-type="fig">7D</a>). Similarly, Klotho treatment inhibited gene expression of β-catenin compared with untreated control cells (*P* \< 0.05; *P* = 0.023) (Figure <a href="#F7" data-ref-type="fig">7C</a>). Real-time PCR analysis also indicated that a concomitant increase in Klotho mRNA levels occurred when nucleus pulposus cells were treated with exogenous Klotho (*P* \< 0.05; *P* = 0.028) (Figure <a href="#F7" data-ref-type="fig">7C</a>). These findings indicate that Wnt signaling is inhibited by Klotho in nucleus pulposus cells.

# Discussion

In this study, we focused on Wnt signaling to clarify the mechanism (upstream or downstream) underlying intervertebral disc degeneration. First, we examined the relation between Wnt signaling and the oxemic status of nucleus pulposus cells. Nucleus pulposus is an avascular tissue, and in some species, pO~2~ levels in large discs may be low. Oxygen tension is a powerful stimulus of nucleus pulposus cells, yet the net result of hypoxia--anabolic or catabolic--in nucleus pulposus cells is not known. In addition, the mechanism by which the nucleus pulposus cells survive in the hypoxic environment of the intervertebral disc remains largely unknown, and no evidence suggests how oxygen levels change with disc degeneration *in vivo*. However, *in vitro* studies demonstrate stimulatory effects of hypoxia on nucleus pulposus proliferation and matrix synthesis \[35,36\]. It is very difficult to conclude that low oxygen would be bad for discs from all of these study data, because the existence of complex signaling networks is found in disc degeneration. However, in this study, we found a hypoxia-dependent increase in the expression of β-catenin and a concomitant increase in Wnt signaling activity. We demonstrated previously that activation of Wnt signaling elevates the gene expression and activity of aggrecan by the balance of catabolic and anabolic factors in nucleus pulposus cells. This is the key to understanding the biology of intervertebral disc maintenance and degeneration \[26\]. Mwale *et al.* \[37\] reported that low oxygen levels increase aggrecan mRNA expression in nucleus pulposus cells and that disc cell metabolism is not impaired at low oxygen concentrations. Our results indicate that effect of hypoxia on matrix synthesis of the nucleus pulposus cells might be directly mediated by Wnt signaling or indirectly by Wnt stimulation of other pathways, such as the hypoxia-inducible factor (HIF) pathway. However, this study was unable to distinguish whether this is a direct or an indirect effect. Kaidi *et al.* \[38\] reported that Wnt signaling is mediated by the physical interaction between HIF-1α and β-catenin. Intriguingly, the β-catenin/HIF-1α interaction increases HIF transcriptional activity, which might help cells adapt to severe hypoxia. Risbud *et al.* \[39\] reported that nucleus pulposus cells display robust and constitutive expression of both HIF-1α and HIF-2α, suggesting that these cells reside in a hypoxic environment. Future studies will address the role of the β-catenin/HIF pathways to determine whether regulation of Wnt signaling by hypoxic conditions is specific to nucleus pulposus cells.

Second, we investigated the expression of Klotho, a newly identified antiaging gene, and examined the relation in nucleus pulposus cells of Wnt signaling and Klotho.

Finally, we investigated the role of Klotho on aggrecan or collagen type-II expression and the proliferation of nucleus pulposus cells.

It is clear that the incidence of disc degeneration increases markedly with age. Several studies have focused on Klotho and its role in the aging phenotype. The Klotho protein is composed of a large extracellular domain (130 kDa), a transmembrane domain, and a very short intracellular domain (10 amino acids). The entire extracellular domain is released into the extracellular space and is detectable in blood, urine, and cerebrospinal fluid \[40\]. Membrane Klotho functions as a receptor for a hormone that regulates excretion of phosphate and synthesis of active vitamin D in the kidney \[41,42\]. Secreted Klotho regulates the activity of multiple growth factors, including insulin/insulin-like growth factor-1 (IGF-1) \[11\], Wnt \[28\], and transforming growth factor (TGF)-β1 \[43\]. Liu *et al.* \[28\] demonstrated that Klotho acts as a Wnt antagonist and suggested that prolonged Wnt stimulation may contribute to stem cell depletion and aging. They reported that secreted Klotho inhibits Wnt signaling activity by binding directly to Wnt ligands and preventing them from binding to their receptors. In this study, we performed molecular functional analysis to study the expression of Klotho in the intervertebral disc and to elucidate the signal crosstalk between Klotho and Wnt signaling; this crosstalk is attracting attention as a potential trigger for degeneration in the intervertebral disc. Interestingly, we demonstrated for the first time that the transcriptional activation of Klotho is associated with promoter demethylation by Wnt signaling. Accordingly, these experiments strongly suggested that in nucleus pulposus cells, activation of Wnt signaling induces an increase in Klotho promoter activity and gene and protein expression by demethylation. By contrast, activation of nucleus pulposus cells by secreted Klotho seems to suppress Wnt signaling. These results might support that Klotho is not simply an antagonist of Wnt signaling but that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells.

We hypothesized that Wnt signaling might control nucleus pulposus homeostasis in normal intervertebral discs. However, although we know that, in general, Klotho is an antiaging gene, activation of Wnt signaling nevertheless resulted in induction of Klotho in nucleus pulposus cells. Consequently, Klotho inhibited aggrecan synthesis and proliferation of nucleus pulposus cells. This is unexpected, because suppression of aggrecan synthesis and proliferation of nucleus pulposus cells would appear to be potentially damaging to the intervertebral disc. The reason for these discrepant effects is not clear, but several possible explanations exist.

First, it is possible that Klotho interacts with signal pathways other than Wnt signaling. Klotho can affect the function of Wnt, TGF-β1, and IGF, which can affect the expression of aggrecan and collagen type II in different ways. Doi *et al.* \[43\] reported that secreted Klotho suppresses TGF-β1 signaling by directly binding to type-II TGF-β receptor (TGFβR2) on the cell surface and preventing TGF-β1 binding to that receptor. TGF-β1 is the most potent inducer of matrix synthesis in nucleus pulposus cells. In addition, fibroblast growth factor (FGF) may act as a catabolic and antianabolic mediator in nucleus pulposus cells, stimulating matrix metalloproteinase (MMP)-13 expression and suppressing proteoglycan synthesis. Recently, Kuro-o \[44\] reported that the most important function of the Klotho family is to regulate the function of the endocrine FGF family. Therefore, the final effect of Klotho on the aggrecan and collagen type II gene expression may be the result of the different effects.

Second, these mechanisms may be specific to the nucleus pulposus cells, because this cell is unique both embryologically and functionally. Moreover, although regulation of Wnt signaling may be tissue specific, the signaling pathway has been shown to modulate cellular proliferation or differentiation.

Third, these differences may relate to the age and species of the animal from which the cells are isolated and the environment in which the cell metabolism was studied. Other groups have shown that glycosaminoglycan production by nucleus pulposus and annulus fibrosus cells can vary, depending on the source of disc cells \[45\].

Fourth, the *Klotho* gene encodes several isoforms in mammals, which include a membrane-bound and two secreted forms. Therefore, the results for this study might be from the different isoforms through different signaling pathways.

Future studies will address the role of Wnt signaling by using human samples to determine whether the regulation of cell growth and matrix synthesis by Klotho is specific to nucleus pulposus cells. Moreover, it is necessary to examine whether, in nucleus pulposus cells, Klotho affects the activity of several signaling pathways, including those of TGF, FGF, or the MMP family, which may participate in intervertebral disc degeneration. Wnt signals are regulated in various ways in response to various extracellular and intracellular signaling. However, DKK, which regulates extracellular Wnt signaling, did not inhibit the reporter activation of Klotho in this study. The DKK family comprises four subtypes, which affect Wnt signaling by combining with the collaboration receptor LRP5/6 of each Wnt-Fzd \[46,47\]. The subtypes seem to influence the functional differences and types of cells that develop, although it is generally believed that only DKK1 and DKK4 inhibit Wnt signals. Our present results suggest that the action of the regulatory factor DKK outside of cells is not dependent on the expression of Klotho in nucleus pulposus cells.

# Conclusions

Here, we demonstrated that (1) hypoxia stimulates Wnt signaling in nucleus pulposus cells; (2) Klotho is expressed in intervertebral discs; (3) transcriptional activation of Klotho is associated with promoter demethylation by Wnt signaling; and (4) Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. In summary, our study demonstrates that in nucleus pulposus cells, the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling. To elucidate the mechanism causing intervertebral disc degeneration, further studies are needed to determine how the interplay of various Wnt ligands and Klotho modulates cell growth and matrix synthesis in nucleus pulposus cells.

# Abbreviations

BGS: bisulfite genomic sequencing; BIO: 6-bromoindirubin-3'-oxime; CPG: cytosine-phosphate-guanosine; DKK: Dickkopf; HIF-1α: hypoxia-inducible factor 1α; TCF/LEF: T-cell factor/lymphoid enhancer factor.

# Competing interests

The authors declare that they have no competing interests.

# Authors' contributions

AH and FA made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. AH, FA, KY, DS, and JM were involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved the manuscript for publication.

# Supplementary Material

###### Additional file 1

**Nucleus pulposus cells were cotransfected with the Klotho reporter plasmid along with 500 ng of Dkk1, Dkk2, Dkk3, Dkk4, or the empty backbone vector, and the reporter activity was measured (left panel)**. Nucleus pulposus cells were cotransfected with the Klotho promoter plasmid along with increasing quantities (100 to 500 ng) of the Dkk1 expression plasmid or with the empty backbone vector, and the reporter activity was measured (right panel). The results were normalized for transfection efficiency and are expressed as a relative ratio of luciferase to pGL4.74 activities (denoted as relative activity). \**P \< 0.05* between groups. Error bars represent SDs. NS, not significant.

Click here for file

## Acknowledgements

We greatly appreciate the helpful advice of Dr. Tadayuki Sato and the technical assistance of Hideki Hayashi, Masayuki Tanaka, and Mayumi Nakano. This study was supported in part by the Research and Study Program of Tokai University Educational System General Research Organization and Grant-in -Aid for Scientific Research (KAKENHI)