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1.  Endovascular Radiofrequency Ablation for Varicose Veins 
Executive Summary
Objective
The objective of the MAS evidence review was to conduct a systematic review of the available evidence on the safety, effectiveness, durability and cost–effectiveness of endovascular radiofrequency ablation (RFA) for the treatment of primary symptomatic varicose veins.
Background
The Ontario Health Technology Advisory Committee (OHTAC) met on August 26th, 2010 to review the safety, effectiveness, durability, and cost-effectiveness of RFA for the treatment of primary symptomatic varicose veins based on an evidence-based review by the Medical Advisory Secretariat (MAS).
Clinical Condition
Varicose veins (VV) are tortuous, twisted, or elongated veins. This can be due to existing (inherited) valve dysfunction or decreased vein elasticity (primary venous reflux) or valve damage from prior thrombotic events (secondary venous reflux). The end result is pooling of blood in the veins, increased venous pressure and subsequent vein enlargement. As a result of high venous pressure, branch vessels balloon out leading to varicosities (varicose veins).
Symptoms typically affect the lower extremities and include (but are not limited to): aching, swelling, throbbing, night cramps, restless legs, leg fatigue, itching and burning. Left untreated, venous reflux tends to be progressive, often leading to chronic venous insufficiency (CVI). A number of complications are associated with untreated venous reflux: including superficial thrombophlebitis as well as variceal rupture and haemorrhage. CVI often results in chronic skin changes referred to as stasis dermatitis. Stasis dermatitis is comprised of a spectrum of cutaneous abnormalities including edema, hyperpigmentation, eczema, lipodermatosclerosis and stasis ulceration. Ulceration represents the disease end point for severe CVI. CVI is associated with a reduced quality of life particularly in relation to pain, physical function and mobility. In severe cases, VV with ulcers, QOL has been rated to be as bad or worse as other chronic diseases such as back pain and arthritis.
Lower limb VV is a very common disease affecting adults – estimated to be the 7th most common reason for physician referral in the US. There is a very strong familial predisposition to VV. The risk in offspring is 90% if both parents affected, 20% when neither affected and 45% (25% boys, 62% girls) if one parent affected. The prevalence of VV worldwide ranges from 5% to 15% among men and 3% to 29% among women varying by the age, gender and ethnicity of the study population, survey methods and disease definition and measurement. The annual incidence of VV estimated from the Framingham Study was reported to be 2.6% among women and 1.9% among men and did not vary within the age range (40-89 years) studied.
Approximately 1% of the adult population has a stasis ulcer of venous origin at any one time with 4% at risk. The majority of leg ulcer patients are elderly with simple superficial vein reflux. Stasis ulcers are often lengthy medical problems and can last for several years and, despite effective compression therapy and multilayer bandaging are associated with high recurrence rates. Recent trials involving surgical treatment of superficial vein reflux have resulted in healing and significantly reduced recurrence rates.
Endovascular Radiofrequency Ablation for Varicose Veins
RFA is an image-guided minimally invasive treatment alternative to surgical stripping of superficial venous reflux. RFA does not require an operating room or general anaesthesia and has been performed in an outpatient setting by a variety of medical specialties including surgeons and interventional radiologists. Rather than surgically removing the vein, RFA works by destroying or ablating the refluxing vein segment using thermal energy delivered through a radiofrequency catheter.
Prior to performing RFA, color-flow Doppler ultrasonography is used to confirm and map all areas of venous reflux to devise a safe and effective treatment plan. The RFA procedure involves the introduction of a guide wire into the target vein under ultrasound guidance followed by the insertion of an introducer sheath through which the RFA catheter is advanced. Once satisfactory positioning has been confirmed with ultrasound, a tumescent anaesthetic solution is injected into the soft tissue surrounding the target vein along its entire length. This serves to anaesthetize the vein, insulate the heat from damaging adjacent structures, including nerves and skin and compresses the vein increasing optimal contact of the vessel wall with the electrodes or expanded prongs of the RF device. The RF generator is then activated and the catheter is slowly pulled along the length of the vein. At the end of the procedure, hemostasis is then achieved by applying pressure to the vein entry point.
Adequate and proper compression stockings and bandages are applied after the procedure to reduce the risk of venous thromboembolism and to reduce postoperative bruising and tenderness. Patients are encouraged to walk immediately after the procedure. Follow-up protocols vary, with most patients returning 1 to 3 weeks later for an initial follow-up visit. At this point, the initial clinical result is assessed and occlusion of the treated vessels is confirmed with ultrasound. Patients often have a second follow-up visit 1 to 3 months following RFA at which time clinical evaluation and ultrasound are repeated. If required, additional procedures such as phlebectomy or sclerotherapy may be performed during the RFA procedure or at any follow-up visits.
Regulatory Status
The Closure System® radiofrequency generator for endovascular thermal ablation of varicose veins was approved by Health Canada as a class 3 device in March 2005, registered under medical device license 67865. The RFA intravascular catheter was approved by Health Canada in November 2007 for the ClosureFast catheter, registered under medical device license 16574. The Closure System® also has regulatory approvals in Australia, Europe (CE Mark) and the United States (FDA clearance). In Ontario, RFA is not an insured service and is currently being introduced in private clinics.
Methods
Literature Search
The MAS evidence–based review was performed to support public financing decisions. The literature search was performed on March 9th, 2010 using standard bibliographic databases for studies published up until March, 2010.
Inclusion Criteria
English language full-reports and human studies Original reports with defined study methodologyReports including standardized measurements on outcome events such as technical success, safety, effectiveness, durability, quality of life or patient satisfaction Reports involving RFA for varicose veins (great or small saphenous veins)Randomized controlled trials (RCTs), systematic reviews and meta-analysesCohort and controlled clinical studies involving ≥ 1 month ultrasound imaging follow-up
Exclusion Criteria
Non systematic reviews, letters, comments and editorials Reports not involving outcome events such as safety, effectiveness, durability, or patient satisfaction following an intervention with RFAReports not involving interventions with RFA for varicose veinsPilot studies or studies with small samples (< 50 subjects)
Summary of Findings
The MAS evidence search on the safety and effectiveness of endovascular RFA ablation of VV identified the following evidence: three HTAs, nine systematic reviews, eight randomized controlled trials (five comparing RFA to surgery and three comparing RFA to ELT), five controlled clinical trials and fourteen cohort case series (four were multicenter registry studies).
The majority (12⁄14) of the cohort studies (3,664) evaluating RFA for VV involved treatment with first generation RFA catheters and the great saphenous vein (GSV) was the target vessel in all studies. Major adverse events were uncommonly reported and the overall pooled major adverse event rate extracted from the cohort studies was 2.9% (105⁄3,664). Imaging defined treatment effectiveness of vein closure rates were variable ranging from 68% to 96% at post-operative follow-up. Vein ablation rate at 6-month follow-up was reported in four studies with rates close to 90%. Only one study reported vein closure rates at 2 years but only for a minority of the eligible cases. The two studies reporting on RFA ablation with the more efficient second generation catheters involved better follow-up and reported higher ablation rates close to 100% at 6-month follow-up with no major adverse events. A large prospective registry trial that recruited over 1,000 patients at thirty-four largely European centers reported on treatment success in six overlapping reports on selected patient subgroups at various follow-up points up to 5 year. However, the follow-up for eligible recruited patients at all time points was low resulting in inadequate estimates of longer term treatment efficacy.
The overall level of evidence of randomized trials comparing RFA with surgical ligation and vein stripping (n = 5) was graded as low to moderate. In all trials RFA ablation was performed with first generation catheters in the setting of the operating theatre under general anaesthesia, usually without tumescent anaesthesia. Procedure times were significantly longer after RFA than surgery. Recovery after treatment was significantly quicker after RFA both with return to usual activity and return to work with on average a one week less of work loss. Major adverse events occurring after surgery were higher [(1.8% (n=4) vs. 0.4% (n = 1) than after RFA but not significantly. Treatment effectiveness measured by imaging defined vein absence or vein closure was comparable in the two treatment groups. Significant improvements in vein symptoms and quality of life over baseline were reported for both treatment groups. Improvements in these outcomes were significantly greater in the RFA group than the surgery group in the peri-operative period but not in later follow-up. Follow-up in these trials was inadequate to evaluate longer term recurrence for either treatment. Patient satisfaction was reported to be high for both treatments but was higher for RFA.
The studies comparing endovascular treatment approaches for VV (RFA and ELT) were more limited. Three RCT studies compared RFA (two with the second generation catheter) with ELT but mainly focused on peri-procedural outcomes such as pain, complications and recovery. Vein ablation rates were not evaluated in the trials, except for one small trial involving bilateral VV. Pain responses in patients undergoing ablation were extremely variable and up to 2 weeks, mean pain levels were significantly less with RFA than ELT ablation but differences were not significant at one month. Recovery, evaluated as return to usual activity or return to work, however, was similar in the treatment groups. Vein symptom and QOL improvements were improved in both groups but were significantly better in the RFA group than the ELT group at 2 weeks, but not at one month. Vein ablation rates were evaluated in several controlled clinical studies comparing the treatments between centers or within centers between individuals or over time. Comparisons in these studies were inconsistent with vein ablation rates for RFA reported to be similar to, higher than and lower than those with ELT.
Economic Analysis
RFA and surgical vein stripping, the main comparator reimbursed by the public system, are comparable in clinical benefits. Hence a cost-analysis was conducted to identify the differences in resources and costs between both procedures and a budgetary impact analysis (BIA) was conducted to project costs over a 5- year period in the province of Ontario. The target population of this economic analysis was patients with symptomatic varicose veins and the primary analytic perspective was that of the Ministry of Health and Long-Term Care.
The average case cost (based on Ontario hospital costs and medical resources) for surgical vein stripping was estimated to be $1,799. In order to calculate a procedural cost for RFA it was assumed that the hospital cost and physician labour fees, excluding anaesthesia and surgical assistance, were the same as vein stripping surgery. The manufacturer also provided details on the generator with a capital cost of $27,500 and a lifespan of 5 years and the disposables (catheter, sheath, guidewire) with a cost of $673 per case. The average case cost for RFA was therefore estimated to be $1,356. One-way sensitivity analysis was also conducted with hospital cost of RFA varied to 60% that of vein stripping surgery (average cost per case = $627.08) to calculate an impact to the province.
Historical volumes of vein stripping surgeries in Ontario were used to project surgeries in a linear fashion up to five years into the future. Volumes for RFA and ELT were calculated based on share capture from the surgery market based on discussion with clinical expert opinion and existing private data based on discussion with the manufacturer. RFA is expected to compete with ELT and capture some of the market. If ELT is reimbursed by the public sector then numbers will continue to increase from previous private data and share capture from the conventional surgical treatment market. Therefore, RFA cases will also increase since it will be capturing a share of the ELT market. A budget impact to the province was then calculated by multiplying volumes by the cost of the procedure.
RFA is comparable in clinical benefits to vein stripping surgery. It has the extra upfront cost of the generator and cost per case for disposables but does not require an operating theater, anaesthetist or surgical assistant fees. The impact to the province is expected to be 5 M by Year 5 with the introduction of new ELT and RFA image guided endovascular technologies and existing surgery for varicose veins.
Conclusion
The conclusions on the comparative outcomes between endovascular RFA and surgical ligation and saphenous vein stripping and between endovascular RFA and laser ablation for VV treatment are summarized in the table below (ES Table 1).
Outcome comparisons of RFA vs. surgery and RFA vs ELT for varicose veins
ELT refers to endovascular laser ablation; RFA, radiofrequency ablation
The outcomes of the evidence-based review on these treatments for VV based on different perspectives are summarized below:
RFA First versus Second Generation Catheters and Segmental Ablation
Ablation with second generation catheters and segmental ablation offered technical advantages with improved ease and significant decreases in procedure time. RFA ablation with second generation catheters is also no longer restricted to smaller (< 12 mm diameter) saphenous veins. The safety profile with the new device and method of energy delivery is as good as or improved over the first generation device. No major adverse events were reported in two multicenter prospective cohort studies in 6 month follow-up with over 500 patients. Post-operative complications such as bruising and pain were significantly less with RFA ablation with second generation catheters than ELT in two RCT trials.RFA treatment with second generation catheters has ablation rates that are higher than with first generation catheters and are more comparable with the consistently high rates of ELT.
Endovascular RFA versus Surgery
RFA has a quicker recovery attributable to decreased pain and lower minor complications.RFA, in the short term was comparable to surgery in treatment effectiveness as assessed by imaging defined anatomic outcomes such as vein closure, flow or reflux. Other treatment outcomes such as symptomatic relief and HRQOL were significantly improved in both groups and between group differences in the early peri-operative period were likely influenced by pain experiences. Longer term follow-up was inadequate to evaluate recurrence after either treatment.Patient satisfaction was high after both treatments but was higher for RFA than surgery.
Endovascular RFA versus ELT
RFA has significantly less post-operative pain than ELT but differences were not significant when pain was adjusted for analgesic use and pain differences between groups did not persist at 1 month follow-up.Treatment effectiveness, measured as symptom relief and QOL improvement were similar between the endovascular treatments in the short term (within 1 month) Treatment effectiveness measured as imaging defined vein ablation was not measured in any RCT trials (only for bilateral VV disease) and results were inconsistently reported in observational trials.Longer term follow-up was not available to assess recurrence after either treatment.
System Outcomes – RFA Replacing Surgery or Competing with ELT
RFA may offer system advantages in that the treatment can be offered by several medical specialties in outpatient settings and because it does not require an operating theatre or general anaesthesia. The treatment may result in decanting of patients from OR with decreased pre-surgical investigations, demand on anaesthetists’ time, hospital stay and wait time for VV treatment. It may also provide more reliable outpatient scheduling. Procedure costs may be less for endovascular approaches than surgery but the budget impact may be greater with insurance of RFA because of the transfer of cases from the private market to the public payer system.Competition between RFA and ELT endovascular approaches is likely to continue to stimulate innovation and technical changes to advance patient care and result in competitive pricing.
PMCID: PMC3377553  PMID: 23074413
2.  Regulation of glucose homeostasis in humans with denervated livers. 
Journal of Clinical Investigation  1997;100(4):931-941.
The liver plays a major role in regulating glucose metabolism, and since its function is influenced by sympathetic/ parasympathetic innervation, we used liver graft as a model of denervation to study the role of CNS in modulating hepatic glucose metabolism in humans. 22 liver transplant subjects were randomly studied by means of the hyperglycemic/ hyperinsulinemic (study 1), hyperglycemic/isoinsulinemic (study 2), euglycemic/hyperinsulinemic (study 3) as well as insulin-induced hypoglycemic (study 4) clamp, combined with bolus-continuous infusion of [3-3H]glucose and indirect calorimetry to determine the effect of different glycemic/insulinemic levels on endogenous glucose production and on peripheral glucose uptake. In addition, postabsorptive glucose homeostasis was cross-sectionally related to the transplant age (range = 40 d-35 mo) in 4 subgroups of patients 2, 6, 15, and 28 mo after transplantation. 22 subjects with chronic uveitis (CU) undergoing a similar immunosuppressive therapy and 35 normal healthy subjects served as controls. The results showed that successful transplantation was associated with fasting glucose concentration and endogenous glucose production in the lower physiological range within a few weeks after transplantation, and this pattern was maintained throughout the 28-mo follow-up period. Fasting glucose (4. 55+/-0.06 vs. 4.75+/-0.06 mM; P = 0.038) and endogenous glucose production (11.3+/-0.4 vs. 12.9+/-0.5 micromol/[kg.min]; P = 0.029) were lower when compared to CU and normal patients. At different combinations of glycemic/insulinemic levels, liver transplant (LTx) patients showed a comparable inhibition of endogenous glucose production. In contrast, in hypoglycemia, after a temporary fall endogenous glucose production rose to values comparable to those of the basal condition in CU and normal subjects (83+/-5 and 92+/-5% of basal), but it did not in LTx subjects (66+/-7%; P < 0.05 vs. CU and normal subjects). Fasting insulin and C-peptide levels were increased up to 6 mo after transplantation, indicating insulin resistance partially induced by prednisone. In addition, greater C-peptide but similar insulin levels during the hyperglycemic clamp (study 1) suggested an increased hepatic insulin clearance in LTx as compared to normal subjects. Fasting glucagon concentration was higher 6 mo after transplantation and thereafter. During euglycemia/hyperinsulinemia (study 3), the insulin-induced glucagon suppression detectable in CU and normal subjects was lacking in LTx subjects; furthermore, the counterregulatory response during hypoglycemia was blunted. In summary, liver transplant subjects have normal postabsorptive glucose metabolism, and glucose and insulin challenge elicit normal response at both hepatic and peripheral sites. Nevertheless, (a) minimal alteration of endogenous glucose production, (b) increased concentration of insulin and glucagon, and (c) defective counterregulation during hypoglycemia may reflect an alteration of the liver-CNS-islet circuit which is due to denervation of the transplanted graft.
PMCID: PMC508266  PMID: 9259593
3.  Polysome Profiling in Liver Identifies Dynamic Regulation of Endoplasmic Reticulum Translatome by Obesity and Fasting 
PLoS Genetics  2012;8(8):e1002902.
Obesity-associated metabolic complications are generally considered to emerge from abnormalities in carbohydrate and lipid metabolism, whereas the status of protein metabolism is not well studied. Here, we performed comparative polysome and associated transcriptional profiling analyses to study the dynamics and functional implications of endoplasmic reticulum (ER)–associated protein synthesis in the mouse liver under conditions of obesity and nutrient deprivation. We discovered that ER from livers of obese mice exhibits a general reduction in protein synthesis, and comprehensive analysis of polysome-bound transcripts revealed extensive down-regulation of protein synthesis machinery, mitochondrial components, and bile acid metabolism in the obese translatome. Nutrient availability also plays an important but distinct role in remodeling the hepatic ER translatome in lean and obese mice. Fasting in obese mice partially reversed the overall translatomic differences between lean and obese nonfasted controls, whereas fasting of the lean mice mimicked many of the translatomic changes induced by the development of obesity. The strongest examples of such regulations were the reduction in Cyp7b1 and Slco1a1, molecules involved in bile acid metabolism. Exogenous expression of either gene significantly lowered plasma glucose levels, improved hepatic steatosis, but also caused cholestasis, indicating the fine balance bile acids play in regulating metabolism and health. Together, our work defines dynamic regulation of the liver translatome by obesity and nutrient availability, and it identifies a novel role for bile acid metabolism in the pathogenesis of metabolic abnormalities associated with obesity.
Author Summary
Chronic diseases including obesity and associated metabolic abnormalities have become the greatest threat to human health worldwide. How metabolic organs and organelles adapt to nutritional fluctuations, or fail to do so, remains incompletely understood. To explore these issues, we developed a new platform to explore translational responses in the liver, a critical organ for metabolic homeostasis. In this translatomic platform, we integrated polysome profiling and global analysis of polysome-associated mRNAs to systematically quantify protein synthesis on each transcript in obesity and during fasting. Our analysis demonstrated for the first time that protein synthesis is progressively suppressed in the obese liver and that the overall translatome profile of obese liver markedly resembles that of fasting lean mice, particularly in mitochondrial function and bile metabolism. We also examined the physiological impact of some of these alterations and concluded that aberrant bile acid metabolism in the obese liver represents a novel mechanism contributing to hyperglycemia and continuous weight gain. Together, our work reveals abnormal translational regulation as a novel aspect of obesity that could impact future directions in metabolic disease treatment, and we believe translatome profiling represents a new approach to unravel complex mechanisms regulating cellular function and disease pathology.
doi:10.1371/journal.pgen.1002902
PMCID: PMC3426552  PMID: 22927828
4.  Quantifying the Contribution of the Liver to Glucose Homeostasis: A Detailed Kinetic Model of Human Hepatic Glucose Metabolism 
PLoS Computational Biology  2012;8(6):e1002577.
Despite the crucial role of the liver in glucose homeostasis, a detailed mathematical model of human hepatic glucose metabolism is lacking so far. Here we present a detailed kinetic model of glycolysis, gluconeogenesis and glycogen metabolism in human hepatocytes integrated with the hormonal control of these pathways by insulin, glucagon and epinephrine. Model simulations are in good agreement with experimental data on (i) the quantitative contributions of glycolysis, gluconeogenesis, and glycogen metabolism to hepatic glucose production and hepatic glucose utilization under varying physiological states. (ii) the time courses of postprandial glycogen storage as well as glycogen depletion in overnight fasting and short term fasting (iii) the switch from net hepatic glucose production under hypoglycemia to net hepatic glucose utilization under hyperglycemia essential for glucose homeostasis (iv) hormone perturbations of hepatic glucose metabolism. Response analysis reveals an extra high capacity of the liver to counteract changes of plasma glucose level below 5 mM (hypoglycemia) and above 7.5 mM (hyperglycemia). Our model may serve as an important module of a whole-body model of human glucose metabolism and as a valuable tool for understanding the role of the liver in glucose homeostasis under normal conditions and in diseases like diabetes or glycogen storage diseases.
Author Summary
Glucose is an indispensable fuel for all cells and organs, but at the same time leads to problems at high concentrations. As a consequence, blood glucose is controlled in a narrow range to guarantee constant supply and on the other hand avoid damages associated with elevated glucose levels. The liver is the main organ controlling blood glucose by (i) releasing newly synthesized or stored glucose in the blood stream when blood glucose is low (ii) using and storing glucose when blood glucose is elevated. These processes are regulated by hormones, in particular insulin, glucagon and epinephrine. We developed the first detailed kinetic model of this crucial metabolic system integrated with its hormonal control and validated the model based on a multitude of experimental data. Our model enables for the first time to simulate hepatic glucose metabolism in depth. Our results show how due to the hormonal control of key enzymes the liver metabolism can be switched between glucose production and utilization. We provide an essential model to analyze glucose regulation in the normal state and diseases associated with defects in glucose homeostasis like diabetes.
doi:10.1371/journal.pcbi.1002577
PMCID: PMC3383054  PMID: 22761565
5.  Endovascular Laser Therapy for Varicose Veins 
Executive Summary
Objective
The objective of the MAS evidence review was to conduct a systematic review of the available evidence on the safety, effectiveness, durability and cost–effectiveness of endovascular laser therapy (ELT) for the treatment of primary symptomatic varicose veins (VV).
Background
The Ontario Health Technology Advisory Committee (OHTAC) met on November 27, 2009 to review the safety, effectiveness, durability and cost-effectiveness of ELT for the treatment of primary VV based on an evidence-based review by the Medical Advisory Secretariat (MAS).
Clinical Condition
VV are tortuous, twisted, or elongated veins. This can be due to existing (inherited) valve dysfunction or decreased vein elasticity (primary venous reflux) or valve damage from prior thrombotic events (secondary venous reflux). The end result is pooling of blood in the veins, increased venous pressure and subsequent vein enlargement. As a result of high venous pressure, branch vessels balloon out leading to varicosities (varicose veins).
Symptoms typically affect the lower extremities and include (but are not limited to): aching, swelling, throbbing, night cramps, restless legs, leg fatigue, itching and burning. Left untreated, venous reflux tends to be progressive, often leading to chronic venous insufficiency (CVI).
A number of complications are associated with untreated venous reflux: including superficial thrombophlebitis as well as variceal rupture and haemorrhage. CVI often results in chronic skin changes referred to as stasis dermatitis. Stasis dermatitis is comprised of a spectrum of cutaneous abnormalities including edema, hyperpigmentation, eczema, lipodermatosclerosis and stasis ulceration. Ulceration represents the disease end point for severe CVI.
CVI is associated with a reduced quality of life particularly in relation to pain, physical function and mobility. In severe cases, VV with ulcers, QOL has been rated to be as bad or worse as other chronic diseases such as back pain and arthritis.
Lower limb VV is a common disease affecting adults and estimated to be the seventh most common reason for physician referral in the US. There is a strong familial predisposition to VV with the risk in offspring being 90% if both parents affected, 20% when neither is affected, and 45% (25% boys, 62% girls) if one parent is affected. Globally, the prevalence of VV ranges from 5% to 15% among men and 3% to 29% among women varying by the age, gender and ethnicity of the study population, survey methods and disease definition and measurement. The annual incidence of VV estimated from the Framingham Study was reported to be 2.6% among women and 1.9% among men and did not vary within the age range (40-89 years) studied.
Approximately 1% of the adult population has a stasis ulcer of venous origin at any one time with 4% at risk. The majority of leg ulcer patients are elderly with simple superficial vein reflux. Stasis ulcers are often lengthy medical problems and can last for several years and, despite effective compression therapy and multilayer bandaging are associated with high recurrence rates. Recent trials involving surgical treatment of superficial vein reflux have resulted in healing and significantly reduced recurrence rates.
Endovascular Laser Therapy for VV
ELT is an image-guided, minimally invasive treatment alternative to surgical stripping of superficial venous reflux. It does not require an operating room or general anesthesia and has been performed in outpatient settings by a variety of medical specialties including surgeons (vascular or general), interventional radiologists and phlebologists. Rather than surgically removing the vein, ELT works by destroying, cauterizing or ablating the refluxing vein segment using heat energy delivered via laser fibre.
Prior to ELT, colour-flow Doppler ultrasonography is used to confirm and map all areas of venous reflux to devise a safe and effective treatment plan. The ELT procedure involves the introduction of a guide wire into the target vein under ultrasound guidance followed by the insertion of an introducer sheath through which an optical fibre carrying the laser energy is advanced. A tumescent anesthetic solution is injected into the soft tissue surrounding the target vein along its entire length. This serves to anaesthetize the vein so that the patient feels no discomfort during the procedure. It also serves to insulate the heat from damaging adjacent structures, including nerves and skin. Once satisfactory positioning has been confirmed with ultrasound, the laser is activated. Both the laser fibre and the sheath are simultaneously, slowly and continuously pulled back along the length of the target vessel. At the end of the procedure, homeostasis is then achieved by applying pressure to the entry point.
Adequate and proper compression stockings and bandages are applied after the procedure to reduce the risk of venous thromboembolism, and to reduce postoperative bruising and tenderness. Patients are encouraged to walk immediately after the procedure and most patients return to work or usual activity within a few days. Follow-up protocols vary, with most patients returning 1-3 weeks later for an initial follow-up visit. At this point, the initial clinical result is assessed and occlusion of the treated vessels is confirmed with ultrasound. Patients often have a second follow-up visit 1-3 months following ELT at which time clinical evaluation and ultrasound are repeated. If required, sclerotherapy may be performed during the ELT procedure or at any follow-up visits.
Regulatory Status
Endovascular laser for the treatment of VV was approved by Health Canada as a class 3 device in 2002. The treatment has been an insured service in Saskatchewan since 2007 and is the only province to insure ELT. Although the treatment is not an insured service in Ontario, it has been provided by various medical specialties since 2002 in over 20 private clinics.
Methods
Literature Search
The MAS evidence-based review was performed as an update to the 2007 health technology review performed by the Australian Medical Services Committee (MSAC) to support public financing decisions. The literature search was performed on August 18, 2009 using standard bibliographic databases for studies published from January 1, 2007 to August 15, 2009. Search alerts were generated and reviewed for additional relevant literature up until October 1, 2009.
Inclusion Criteria
English language full-reports and human studies
Original reports with defined study methodology
Reports including standardized measurements on outcome events such as technical success, safety, effectiveness, durability, quality of life or patient satisfaction
Reports involving ELT for VV (great or small saphenous veins)
Randomized controlled trials (RCTs), systematic reviews and meta-analyses
Cohort and controlled clinical studies involving > 1 month ultrasound imaging follow-up
Exclusion Criteria
Non systematic reviews, letters, comments and editorials
Reports not involving outcome events such as safety, effectiveness, durability, or patient satisfaction following an intervention with ELT
Reports not involving interventions with ELT for VV
Pilot studies or studies with small samples ( < 50 subjects)
Summary of Findings
The MAS evidence search identified 14 systematic reviews, 29 cohort studies on safety and effectiveness, four cost studies and 12 randomized controlled trials involving ELT, six of these comparing endovascular laser with surgical ligation and saphenous vein stripping.
Since 2007, 22 cohort studies involving 10,883 patients undergoing ELT of the great saphenous vein (GSV) have been published. Imaging defined treatment effectiveness of mean vein closure rates were reported to be greater than 90% (range 93%- 99%) at short term follow-up. Longer than one year follow-up was reported in five studies with life table analysis performed in four but the follow up was still limited at three and four years. The overall pooled major adverse event rate, including DVT, PE, skin burns or nerve damage events extracted from these studies, was 0.63% (69/10,883).
The overall level of evidence of randomized trials comparing ELT with surgical ligation and vein stripping (n= 6) was graded as moderate to high. Recovery after treatment was significantly quicker after ELT (return to work median number of days, 4 vs. 17; p= .005). Major adverse events occurring after surgery were higher [(1.8% (n=4) vs. 0.4% (n = 1) 1 but not significantly. Treatment effectiveness as measured by imaging vein absence or closure, symptom relief or quality of life similar in the two treatment groups and both treatments resulted in statistically significantly improvements in these outcomes. Recurrence was low after both treatments at follow up but neovascularization (growth of new vessels, a key predictor of long term recurrence was significantly more common (18% vs. 1%; p = .001) after surgery. Although patient satisfaction was reported to be high (>80%) with both treatments, patient preferences evaluated through recruitment process, physician reports and consumer groups were strongly in favour of ELT. For patients minimal complications, quick recovery and dependability of outpatient scheduling were key considerations.
As clinical effectiveness of the two treatments was similar, a cost-analysis was performed to compare differences in resources and costs between the two procedures. A budget impact analysis for introducing ELT as an insured service was also performed. The average case cost (based on Ontario hospital costs and medical resources) for surgical vein stripping was estimated to be $1,799. Because of the uncertainties with resources associated with ELT, in addition to the device related costs, hospital costs were varied and assumed to be the same as or less than (40%) those for surgery resulting in an average ELT case cost of $2,025 or $1,602.
Based on the historical pattern of surgical vein stripping for varices a 5-year projection was made for annual volumes and costs. In Ontario in 2007/2008, 3481 surgical vein stripping procedures were performed, 28% for repeat procedures. Annual volumes of ELT currently being performed in the province in over 20 private clinics were estimated to be approximately 840. If ELT were publicly reimbursed, it was assumed that it would capture 35% of the vein stripping market in the first year and increase to 55% in subsequent years. Based on these assumptions if ELT were not publicly reimbursed, the province would be paying approximately $5.9 million and if ELT were reimbursed the province would pay $8.2 million if the hospital costs for ELT were the same as surgery and $7.1 million if the hospital costs were less (40%) than surgery.
The conclusions on the comparative outcomes between laser ablation and surgical ligation and saphenous vein stripping are summarized in the table below (ES Table 1).
Outcome comparisons of ELT vs. surgery for VV
The outcomes of the evidence-based review on these treatments based on three different perspectives are summarized below:
Patient Outcomes – ELT vs. Surgery
ELT has a quicker recovery attributable to the decreased pain, lower minor complications, use of local anesthesia with immediate ambulation.
ELT is as effective as surgery in the short term as assessed by imaging anatomic outcomes, symptomatic relief and HRQOL outcomes.
Recurrence is similar but neovascularization, a key predictor of long term recurrence, is significantly higher with surgery.
Patient satisfaction is equally high after both treatments but patient preference is much more strongly for ELT. Surgeons performing ELT are satisfied with treatment outcomes and regularly offer ELT as a treatment alternative to surgery.
Clinical or Technical Advantages – ELT Over Surgery
An endovascular approach can more easily and more precisely treat multilevel disease and difficult to treat areas
ELT is an effective and a less invasive treatment for the elderly with VV and those with venous leg ulcers.
System Outcomes – ELT Replacing Surgery
ELT may offer system advantages in that the treatment can be offered by several medical specialties in outpatient settings and because it does not require an operating theatre or general anesthesia.
The treatment may result in ↓ pre-surgical investigations, decanting of patients from OR, ↓ demand on anesthetists time, ↓ hospital stay, ↓decrease wait time for VV treatment and provide more reliable outpatient scheduling.
Depending on the reimbursement mechanism for the treatment, however, it may also result in closure of outpatient clinics with an increasingly centralization of procedures in selected hospitals with large capital budgets resulting in larger and longer waiting lists.
Procedure costs may be similar for the two treatments but the budget impact may be greater with insurance of ELT because of the transfer of the cases from the private market to the public payer system.
PMCID: PMC3377531  PMID: 23074409
6.  Consumption of fructose-sweetened beverages for 10 weeks increases postprandial triacylglycerol and apolipoprotein-B concentrations in overweight and obese women 
The British journal of nutrition  2008;100(5):947-952.
Fructose consumption in the USA has increased over the past three decades. During this time, obesity, insulin resistance and the metabolic syndrome have also increased in prevalence. While diets high in fructose have been shown to promote insulin resistance and increase TAG concentrations in animals, there are insufficient data available regarding the long-term metabolic effects of fructose consumption in humans. The objective of the present study was to investigate the metabolic effects of 10-week consumption of fructose-sweetened beverages in human subjects under energy-balanced conditions in a controlled research setting. Following a 4-week weight-maintaining complex carbohydrate diet, seven overweight or obese (BMI 26.8–33.3 kg/m2) postmenopausal women were fed an isoenergetic intervention diet, which included a fructose-sweetened beverage with each meal, for 10 weeks. The intervention diet provided 15% of energy from protein, 30% from fat and 55% from carbohydrate (30% complex carbohydrate, 25% fructose). Fasting and postprandial glucose, insulin, TAG and apoB concentrations were measured. Fructose consumption increased fasting glucose concentrations and decreased meal-associated glucose and insulin responses (P=0.0002, P=0.007 and P=0.013, respectively). Moreover, after 10 weeks of fructose consumption, 14 h postprandial TAG profiles were significantly increased, with the area under the curve at 10 weeks being 141% higher than at baseline (P=0.04). Fructose also increased fasting apoB concentrations by 19% (P=0.043 v. baseline). In summary, consumption of fructose-sweetened beverages increased postprandial TAG and fasting apoB concentrations, and the present results suggest that long-term consumption of diets high in fructose could lead to an increased risk of CVD.
doi:10.1017/S0007114508968252
PMCID: PMC3038917  PMID: 18384705
Fructose; Glucose; Insulin; Hypertriacylglycerolaemia; Apolipoprotein-B
7.  In vitro reversal of the fasting state of liver metabolism in the rat. Reevaluation of the roles of insulin and glucose. 
Journal of Clinical Investigation  1981;68(1):142-152.
Studies were conducted to determine whether the direction of hepatic carbohydrate and lipid metabolism in the rat could be switched simultaneously from a "fasted" to a "fed" profile in vitro. When incubated for 2 h under appropriate conditions hepatocytes from fasted animals could be induced to synthesize glycogen at in vivo rates. There was concomitant marked elevation of the tissue malonyl-coenzyme A level, acceleration of fatty acid synthesis, and suppression of fatty acid oxidation and ketogenesis. In agreement with reports from some laboratories, but contrary to popular belief, glucose was not taken up efficiently by the cells and was thus a poor substrate for eigher glycogen synthesis or lipogenesis. The best precursor for glycogen formation was fructose, whereas lactate (pyruvate) was most efficient in lipogenesis. In both case the addition of glucose to the gluconeogenic substrates was stimulatory, the highest rates being obtained with the further inclusion of glutamine. Insulin was neither necessary for, nor did it stimulate, glycogen deposition or fatty acid synthesis under favorable substrate conditions. Glucagon at physiological concentrations inhibited both glycogen formation and fatty acid synthesis. Insulin readily reversed the effects of glucagon in the submaximal range of its concentration curve. The following conclusions were drawn. First, the fasted-to-fed transition of hepatic carbohydrate and lipid metabolism can be accomplished in vitro over a time frame similar to that operative in vivo. Second, reversal appears to be a substrate-driven phenomenon, in that insulin is not required. Third, unless an unidentified factor (present in protal blood during feeding) facilitates the uptake of glucose by liver it seems unlikely that glucose is the immediate precursor for liver glycogen or fat synthesis in vivo. A likely candidate for the primary substrate in both processes is lactate, which is rapidly formed from glucose by the small intestine and peripheral tissues. Fructose and amino acids may also contribute. Fourth, the requirement for insulin in the reversal of the fasting state of liver metabolism in vivo can best be explained by its ability to offset the catabolic actions of glucagon.
PMCID: PMC370782  PMID: 7019243
8.  Effects of Dietary Fat and Saturated Fat Content on Liver Fat and Markers of Oxidative Stress in Overweight/Obese Men and Women under Weight-Stable Conditions 
Nutrients  2014;6(11):4678-4690.
Dietary fat and oxidative stress are hypothesized to contribute to non-alcoholic fatty liver disease and progression to steatohepatitis. To determine the effects of dietary fat content on hepatic triglyceride, body fat distribution and markers of inflammation and oxidative stress, overweight/obese subjects with normal glucose tolerance consumed a control diet (CONT: 35% fat/12% saturated fat/47% carbohydrate) for ten days, followed by four weeks on a low fat (LFD (n = 10): 20% fat/8% saturated fat/62% carbohydrate) or high fat diet (HFD (n = 10): 55% fat/25% saturated fat/27% carbohydrate). Hepatic triglyceride content was quantified by MRS and abdominal fat distribution by MRI. Fasting biomarkers of inflammation (plasma hsCRP, IL-6, IL-12, TNFα, IFN-γ) and oxidative stress (urinary F2-α isoprostanes) were measured. Body weight remained stable. Compared to the CONT, hepatic triglyceride decreased on the LFD (mean (95% CI): change −2.13% (−3.74%, −0.52%)), but did not change on the HFD and there was no significant difference between the LFD and HFD. Intra-abdominal fat did not change significantly on either diet, but subcutaneous abdominal fat increased on the HFD. There were no significant changes in fasting metabolic markers, inflammatory markers and urinary F2-α isoprostanes. We conclude that in otherwise healthy overweight/obese adults under weight-neutral conditions, a diet low in fat and saturated fat has modest effects to decrease liver fat and may be beneficial. On the other hand, a diet very high in fat and saturated fat had no effect on hepatic triglyceride or markers of metabolism, inflammation and oxidative stress.
doi:10.3390/nu6114678
PMCID: PMC4245556  PMID: 25353663
non-alcoholic fatty liver; dietary fat; saturated fat; oxidative stress
9.  Metabolic fate of extracted glucose in normal human myocardium. 
Journal of Clinical Investigation  1985;76(5):1819-1827.
Glucose is an important substrate for myocardial metabolism. This study was designed to determine the effect of circulating metabolic substrates on myocardial glucose extraction and to determine the metabolic fate of glucose in normal human myocardium. Coronary sinus and arterial catheters were placed in 23 healthy male volunteers. [6-14C]Glucose was infused as a tracer in 10 subjects. [6-14C]Glucose and [U-13C]lactate were simultaneously infused in the other 13 subjects. Simultaneous blood samples were obtained for chemical analyses of glucose, lactate, and free fatty acids and for the the isotopic analyses of glucose and lactate. Glucose oxidation was assessed by measuring myocardial 14CO2 production. The amount of glucose extracted and oxidized by the myocardium was inversely correlated with the arterial level of free fatty acids (r = -0.71; P less than 0.0001). 20% (range, 0-63%) of the glucose extraction underwent immediate oxidation. Chemical lactate analysis showed a net extraction of 26.0 +/- 16.4%. However, isotopic analysis demonstrated that lactate was being released by the myocardium. In the 13 subjects receiving the dual-carbon-labeled isotopes, the lactate released was 0.09 +/- 0.04 mumol/ml and 49.5 +/- 29.5% of this lactate was from exogenous glucose. This study demonstrates that the circulating level of free fatty acids plays a major role in determining the amount of glucose extracted and oxidized by the normal human myocardium. Only 20.1 +/- 19.4% of the glucose extracted underwent oxidation, and 13.0 +/- 9.0% of the glucose extracted was metabolized to lactate and released by the myocardium. Thus, 60-70% of the glucose extracted by the normal myocardium is probably stored as glycogen in the fasting, resting state.
PMCID: PMC424216  PMID: 4056055
10.  Report of the Committee on the Classification and Diagnostic Criteria of Diabetes Mellitus 
Abstract
Concept of Diabetes Mellitus:
Diabetes mellitus is a group of diseases associated with various metabolic disorders, the main feature of which is chronic hyperglycemia due to insufficient insulin action. Its pathogenesis involves both genetic and environmental factors. The long‐term persistence of metabolic disorders can cause susceptibility to specific complications and also foster arteriosclerosis. Diabetes mellitus is associated with a broad range of clinical presentations, from being asymptomatic to ketoacidosis or coma, depending on the degree of metabolic disorder.
Classification (Tables 1 and 2, and Figure 1):
 Etiological classification of diabetes mellitus and glucose metabolism disorders
Note: Those that cannot at present be classified as any of the above are called unclassifiable.
The occurrence of diabetes‐specific complications has not been confirmed in some of these conditions.
 Diabetes mellitus and glucose metabolism disorders due to other specific mechanisms and diseases
The occurrence of diabetes‐specific complications has not been confirmed in some of these conditions.
 A scheme of the relationship between etiology (mechanism) and patho‐physiological stages (states) of diabetes mellitus. Arrows pointing right represent worsening of glucose metabolism disorders (including onset of diabetes mellitus). Among the arrow lines, indicates the condition classified as ‘diabetes mellitus’. Arrows pointing left represent improvement in the glucose metabolism disorder. The broken lines indicate events of low frequency. For example, in type 2 diabetes mellitus, infection can lead to ketoacidosis and require temporary insulin treatment for survival. Also, once diabetes mellitus has developed, it is treated as diabetes mellitus regardless of improvement in glucose metabolism, therefore, the arrow lines pointing left are filled in black. In such cases, a broken line is used, because complete normalization of glucose metabolism is rare.
The classification of glucose metabolism disorders is principally derived from etiology, and includes staging of pathophysiology based on the degree of deficiency of insulin action. These disorders are classified into four groups: (i) type 1 diabetes mellitus; (ii) type 2 diabetes mellitus; (iii) diabetes mellitus due to other specific mechanisms or diseases; and (iv) gestational diabetes mellitus. Type 1 diabetes is characterized by destruction of pancreatic β‐cells. Type 2 diabetes is characterized by combinations of decreased insulin secretion and decreased insulin sensitivity (insulin resistance). Glucose metabolism disorders in category (iii) are divided into two subgroups; subgroup A is diabetes in which a genetic abnormality has been identified, and subgroup B is diabetes associated with other pathologic disorders or clinical conditions. The staging of glucose metabolism includes normal, borderline and diabetic stages depending on the degree of hyperglycemia occurring as a result of the lack of insulin action or clinical condition. The diabetic stage is then subdivided into three substages: non‐insulin‐ requiring, insulin‐requiring for glycemic control, and insulin‐dependent for survival. The two former conditions are called non‐insulin‐dependent diabetes and the latter is known as insulin‐dependent diabetes. In each individual, these stages may vary according to the deterioration or the improvement of the metabolic state, either spontaneously or by treatment.
Diagnosis (Tables 3–7 and Figure 2):
 Criteria of fasting plasma glucose levels and 75 g oral glucose tolerance test 2‐h value
*Casual plasma glucose ≥200 mg/dL (≥11.1 mmol/L) and HbA1c≥6.5% are also regarded as to indicate diabetic type.
Even for normal type, if 1‐h value is 180 mg/dL (10.0 mmol/L), the risk of progression to diabetes mellitus is greater than for <180 mg/dL (10.0 mmol/L) and should be treated as with borderline type (follow‐up observation, etc.). Fasting plasma glucose level of 100–109 mg/dL (5.5–6.0 mmol/L) is called ‘high‐normal’: within the range of normal fasting plasma glucose.
Plasma glucose level after glucose load in oral glucose tolerance test (OGTT) is not included in casual plasma glucose levels. The value for HbA1c (%) is indicated with 0.4% added to HbA1c (JDS) (%).
 Procedures for diagnosing diabetes mellitus
*The value for HbA1c (%) is indicated with 0.4% added to HbA1c (JDS) (%). **Hyperglycemia must be confirmed in a non‐stressful condition. OGTT, oral glucose tolerance test.
 Disorders and conditions associated with low HbA1c values
 Situations where a 75‐g oral glucose tolerance test is recommended
*The value for HbA1c (%) is indicated with 0.4% added to HbA1c (JDS) (%).
 Definition and diagnostic criteria of gestational diabetes mellitus
(IADPSG Consensus Panel, Reference 42, partly modified with permission of Diabetes Care).
 Flow chart outlining steps in the clinical diagnosis of diabetes mellitus. *The value for HbA1c (%) is indicated with 0.4% added to HbA1c (JDS) (%).
Categories of the State of Glycemia:  Confirmation of chronic hyperglycemia is essential for the diagnosis of diabetes mellitus. When plasma glucose levels are used to determine the categories of glycemia, patients are classified as having a diabetic type if they meet one of the following criteria: (i) fasting plasma glucose level of ≥126 mg/dL (≥7.0 mmol/L); (ii) 2‐h value of ≥200 mg/dL (≥11.1 mmol/L) in 75 g oral glucose tolerance test (OGTT); or (iii) casual plasma glucose level of ≥200 mg/dL (≥11.1 mmol/L). Normal type is defined as fasting plasma glucose level of <110 mg/dL (<6.1 mmol/L) and 2‐h value of <140 mg/dL (<7.8 mmol/L) in OGTT. Borderline type (neither diabetic nor normal type) is defined as falling between the diabetic and normal values. According to the current revision, in addition to the earlier listed plasma glucose values, hemoglobin A1c (HbA1c) has been given a more prominent position as one of the diagnostic criteria. That is, (iv) HbA1c≥6.5% is now also considered to indicate diabetic type. The value of HbA1c, which is equivalent to the internationally used HbA1c (%) (HbA1c [NGSP]) defined by the NGSP (National Glycohemoglobin Standardization Program), is expressed by adding 0.4% to the HbA1c (JDS) (%) defined by the Japan Diabetes Society (JDS).
Subjects with borderline type have a high rate of developing diabetes mellitus, and correspond to the combination of impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) noted by the American Diabetes Association (ADA) and WHO. Although borderline cases show few of the specific complications of diabetes mellitus, the risk of arteriosclerosis is higher than those of normal type. When HbA1c is 6.0–6.4%, suspected diabetes mellitus cannot be excluded, and when HbA1c of 5.6–5.9% is included, it forms a group with a high risk for developing diabetes mellitus in the future, even if they do not have it currently.
Clinical Diagnosis:  1 If any of the criteria for diabetic type (i) through to (iv) is observed at the initial examination, the patient is judged to be ‘diabetic type’. Re‐examination is conducted on another day, and if ‘diabetic type’ is reconfirmed, diabetes mellitus is diagnosed. However, a diagnosis cannot be made only by the re‐examination of HbA1c alone. Moreover, if the plasma glucose values (any of criteria [i], [ii], or [iii]) and the HbA1c (criterion [iv]) in the same blood sample both indicate diabetic type, diabetes mellitus is diagnosed based on the initial examination alone. If HbA1c is used, it is essential that the plasma glucose level (criteria [i], [ii] or [iii]) also indicates diabetic type for a diagnosis of diabetes mellitus. When diabetes mellitus is suspected, HbA1c should be measured at the same time as examination for plasma glucose.2 If the plasma glucose level indicates diabetic type (any of [i], [ii], or [iii]) and either of the following conditions exists, diabetes mellitus can be diagnosed immediately at the initial examination.• The presence of typical symptoms of diabetes mellitus (thirst, polydipsia, polyuria, weight loss)• The presence of definite diabetic retinopathy3 If it can be confirmed that the above conditions 1 or 2 existed in the past, diabetes mellitus can be diagnosed or suspected regardless of the current test results.4 If the diagnosis of diabetes cannot be established by these procedures, the patient is followed up and re‐examined after an appropriate interval.5 The physician should assess not only the presence or absence of diabetes, but also its etiology and glycemic stage, and the presence and absence of diabetic complications or associated conditions.
Epidemiological Study:  For the purpose of estimating the frequency of diabetes mellitus, ‘diabetes mellitus’ can be substituted for the determination of ‘diabetic type’ from a single examination. In this case, HbA1c≥6.5% alone can be defined as ‘diabetes mellitus’.
Health Screening:  It is important not to misdiagnose diabetes mellitus, and thus clinical information such as family history and obesity should be referred to at the time of screening in addition to an index for plasma glucose level.
Gestational Diabetes Mellitus:  There are two hyperglycemic disorders in pregnancy: (i) gestational diabetes mellitus (GDM); and (ii) diabetes mellitus. GDM is diagnosed if one or more of the following criteria is met in a 75 g OGTT during pregnancy:
1 Fasting plasma glucose level of ≥92 mg/dL (5.1 mmol/L)2 1‐h value of ≥180 mg/dL (10.0 mmol/L)3 2‐h value of ≥153 mg/dL (8.5 mmol/L)
However, diabetes mellitus that is diagnosed by the clinical diagnosis of diabetes mellitus defined earlier is excluded from GDM. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00074.x, 2010)
doi:10.1111/j.2040-1124.2010.00074.x
PMCID: PMC4020724  PMID: 24843435
Diabetes mellitus; Clinical diagnosis; HbA1c
11.  HepatoNet1: a comprehensive metabolic reconstruction of the human hepatocyte for the analysis of liver physiology 
We present HepatoNet1, a manually curated large-scale metabolic network of the human hepatocyte that encompasses >2500 reactions in six intracellular and two extracellular compartments.Using constraint-based modeling techniques, the network has been validated to replicate numerous metabolic functions of hepatocytes corresponding to a reference set of diverse physiological liver functions.Taking the detoxification of ammonia and the formation of bile acids as examples, we show how these liver-specific metabolic objectives can be achieved by the variable interplay of various metabolic pathways under varying conditions of nutrients and oxygen availability.
The liver has a pivotal function in metabolic homeostasis of the human body. Hepatocytes are the principal site of the metabolic conversions that underlie diverse physiological functions of the liver. These functions include provision and homeostasis of carbohydrates, amino acids, lipids and lipoproteins in the systemic blood circulation, biotransformation, plasma protein synthesis and bile formation, to name a few. Accordingly, hepatocyte metabolism integrates a vast array of differentially regulated biochemical activities and is highly responsive to environmental perturbations such as changes in portal blood composition (Dardevet et al, 2006). The complexity of this metabolic network and the numerous physiological functions to be achieved within a highly variable physiological environment necessitate an integrated approach with the aim of understanding liver metabolism at a systems level. To this end, we present HepatoNet1, a stoichiometric network of human hepatocyte metabolism characterized by (i) comprehensive coverage of known biochemical activities of hepatocytes and (ii) due representation of the biochemical and physiological functions of hepatocytes as functional network states. The network comprises 777 metabolites in six intracellular (cytosol, endoplasmic reticulum and Golgi apparatus, lysosome, mitochondria, nucleus, and peroxisome) and two extracellular compartments (bile canaliculus and sinusoidal space) and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. We performed flux-balance analyses to validate whether for >300 different metabolic objectives a non-zero stationary flux distribution could be established in the network. Figure 1 shows one such functional flux mode associated with the synthesis of the bile acid glycochenodeoxycholate, one important hepatocyte-specific physiological liver function. Besides those pathways directly linked to the synthesis of the bile acid, the mevalonate pathway and the de novo synthesis of cholesterol, the flux mode comprises additional pathways such as gluconeogenesis, the pentose phosphate pathway or the ornithine cycle because the calculations were routinely performed on a minimal set of exchangeable metabolites, that is all reactants were forced to be balanced and all exportable intermediates had to be catabolized into non-degradable end products. This example shows how HepatoNet1 under the challenges of limited exchange across the network boundary can reveal numerous cross-links between metabolic pathways traditionally perceived as separate entities. For example, alanine is used as gluconeogenic substrate to form glucose-6-phosphate, which is used in the pentose phosphate pathway to generate NADPH. The glycine moiety for bile acid conjugation is derived from serine. Conversion of ammonia into non-toxic nitrogen compounds is one central homeostatic function of hepatocytes. Using the HepatoNet1 model, we investigated, as another example of a complex metabolic objective dependent on systemic physiological parameters, how the consumption of oxygen, glucose and palmitate is affected when an external nitrogen load is converted in varying proportions to the non-toxic nitrogen compounds: urea, glutamine and alanine. The results reveal strong dependencies between the available level of oxygen and the substrate demand of hepatocytes required for effective ammonia detoxification by the liver.
Oxygen demand is highest if nitrogen is exclusively transformed into urea. At lower fluxes into urea, an intriguing pattern for oxygen demand is predicted: oxygen demand attains a minimum if the nitrogen load is directed to urea, glutamine and alanine with relative fluxes of 0.17, 0.43 and 0.40, respectively (Figure 2A). Oxygen demand in this flux distribution is four times lower than for the maximum (100% urea) and still 77 and 33% lower than using alanine and glutamine as exclusive nitrogen compounds, respectively. This computationally predicted tendency is consistent with the notion that the zonation of ammonia detoxification, that is the preferential conversion of ammonia to urea in periportal hepatocytes and to glutamine in perivenous hepatocytes, is dictated by the availability of oxygen (Gebhardt, 1992; Jungermann and Kietzmann, 2000). The decreased oxygen demand in flux distributions using higher proportions of glutamine or alanine is accompanied by increased uptake of the substrates glucose and palmitate (Figure 2B). This is due to an increased demand of energy and carbon for the amidation and transamination of glutamate and pyruvate to discharge nitrogen in the form of glutamine and alanine, respectively. In terms of both scope and specificity, our model bridges the scale between models constructed specifically to examine distinct metabolic processes of the liver and modeling based on a global representation of human metabolism. The former include models for the interdependence of gluconeogenesis and fatty-acid catabolism (Chalhoub et al, 2007), impairment of glucose production in von Gierke's and Hers' diseases (Beard and Qian, 2005) and other processes (Calik and Akbay, 2000; Stucki and Urbanczik, 2005; Ohno et al, 2008). The hallmark of these models is that each of them focuses on a small number of reactions pertinent to the metabolic function of interest embedded in a customized representation of the principal pathways of central metabolism. HepatoNet1, currently, outperforms liver-specific models computationally predicted (Shlomi et al, 2008) on the basis of global reconstructions of human metabolism (Duarte et al, 2007; Ma and Goryanin, 2008). In contrast to either of the aforementioned modeling scales, HepatoNet1 provides the combination of a system-scale representation of metabolic activities and representation of the cell type-specific physical boundaries and their specific transport capacities. This allows for a highly versatile use of the model for the analysis of various liver-specific physiological functions. Conceptually, from a biological system perspective, this type of model offers a large degree of comprehensiveness, whereas retaining tissue specificity, a fundamental design principle of mammalian metabolism. HepatoNet1 is expected to provide a structural platform for computational studies on liver function. The results presented herein highlight how internal fluxes of hepatocyte metabolism and the interplay with systemic physiological parameters can be analyzed with constraint-based modeling techniques. At the same time, the framework may serve as a scaffold for complementation of kinetic and regulatory properties of enzymes and transporters for analysis of sub-networks with topological or kinetic modeling methods.
We present HepatoNet1, the first reconstruction of a comprehensive metabolic network of the human hepatocyte that is shown to accomplish a large canon of known metabolic liver functions. The network comprises 777 metabolites in six intracellular and two extracellular compartments and 2539 reactions, including 1466 transport reactions. It is based on the manual evaluation of >1500 original scientific research publications to warrant a high-quality evidence-based model. The final network is the result of an iterative process of data compilation and rigorous computational testing of network functionality by means of constraint-based modeling techniques. Taking the hepatic detoxification of ammonia as an example, we show how the availability of nutrients and oxygen may modulate the interplay of various metabolic pathways to allow an efficient response of the liver to perturbations of the homeostasis of blood compounds.
doi:10.1038/msb.2010.62
PMCID: PMC2964118  PMID: 20823849
computational biology; flux balance; liver; minimal flux
12.  Insulin resistance in uremia. 
Journal of Clinical Investigation  1981;67(2):563-568.
Tissue sensitivity to insulin was examined with the euglycemic insulin clamp technique in 17 chronically uremic and 36 control subjects. The plasma insulin concentration was raised by approximately 100 microU/ml and the plasma glucose concentration was maintained at the basal level with a variable glucose infusion. Under these steady-state conditions of euglycemia, the glucose infusion rate is a measure of the amount of glucose taken up by the entire body. In uremic subjects insulin-mediated glucose metabolism was reduced by 47% compared with controls (3.71 +/- 0.20 vs. 7.38 +/- 0.26 mg/kg . min; P less than 0.001). Basal hepatic glucose production (measured with [3H]-3-glucose) was normal in uremic subjects (2.17 +/- 0.04 mg/kg . min) and suppressed normally by 94 +/- 2% following insulin administration. In six uremic and six control subjects, net splanchnic glucose balance was also measured directly by the hepatic venous catheterization technique. In the postabsorptive state splanchnic glucose production was similar in uremics (1.57 +/- 0.03 mg/kg . min) and controls (1.79 +/- 0.20 mg/kg . min). After 90 min of sustained hyperinsulinemia, splanchnic glucose balance reverted to a net uptake which was similar in uremics (0.42 +/- 0.11 mg/kg . min) and controls (0.53 +/- 0.12 mg/kg . min). In contrast, glucose uptake by the leg was reduced by 60% in the uremic group (21 +/- 1 vs. 52 +/- 8 mumol/min . kg of leg wt; P less than 0.005) and this decrease closely paralleled the decrease in total glucose metabolism by the entire body. These results indicate that: (a) suppression of hepatic glucose production by physiologic hyperinsulinemia is not impaired by uremia, (b) insulin-mediated glucose uptake by the liver is normal in uremic subjects, and (c) tissue insensitivity to insulin is the primary cause of insulin resistance in uremia.
PMCID: PMC370600  PMID: 7007440
13.  Reducing Liver Fat by Low Carbohydrate Caloric Restriction Targets Hepatic Glucose Production in Non-Diabetic Obese Adults with Non-Alcoholic Fatty Liver Disease 
Journal of clinical medicine  2014;3(3):1050-1063.
Non-alcoholic fatty liver disease (NAFLD) impairs liver functions, the organ responsible for the regulation of endogenous glucose production and thus plays a key role in glycemic homeostasis. Therefore, interventions designed to normalize liver fat content are needed to improve glucose metabolism in patients affected by NAFLD such as obesity.
Objective
this investigation is designed to determine the effects of caloric restriction on hepatic and peripheral glucose metabolism in obese humans with NAFLD.
Methods
eight non-diabetic obese adults were restricted for daily energy intake (800 kcal) and low carbohydrate (<10%) for 8 weeks. Body compositions, liver fat and hepatic glucose production (HGP) and peripheral glucose disposal before and after the intervention were determined.
Results
the caloric restriction reduced liver fat content by 2/3 (p = 0.004). Abdominal subcutaneous and visceral fat, body weight, BMI, waist circumference and fasting plasma triglyceride and free fatty acid concentrations all significantly decreased (p < 0.05). The suppression of post-load HGP was improved by 22% (p = 0.002) whereas glucose disposal was not affected (p = 0.3). Fasting glucose remained unchanged and the changes in the 2-hour plasma glucose and insulin concentration were modest and statistically insignificant (p > 0.05). Liver fat is the only independent variable highly correlated to HGP after the removal of confounders.
Conclusion
NAFLD impairs HGP but not peripheral glucose disposal; low carbohydrate caloric restriction effectively lowers liver fat which appears to directly correct the HGP impairment.
doi:10.3390/jcm3031050
PMCID: PMC4234060  PMID: 25411646
obesity; caloric restriction; liver fat; hepatic glucose production; glucose disposal
14.  The metabolism of circulating maltose in man 
Journal of Clinical Investigation  1971;50(5):986-991.
The utilization of circulating maltose was compared to that of glucose in six normal fasting subjects after intravenous injection of 25 g of either sugar. Blood samples were obtained over a 2 hr period and were assayed for free fatty acids (FFA), insulin, glucose, and total reducing substances. Urine was collected for 2 hr after maltose administration and assayed enzymatically for glucose and maltose. Blood glucose concentrations did not increase after maltose infusion, although a significant rise in total reducing substances was noted, indicating the presence of this disaccharide in the blood. Less than 3% of the administered maltose was excreted in the urine either as maltose or glucose. Initially, there was a fourfold increase in serum insulin concentration after glucose and a threefold increase after maltose infusion. Therefore, serum insulin concentrations gradually declined in a similar manner for both sugars. The plasma FFA at 15 min decreased 371 uEq/liter after glucose and 338 uEq/liter after maltose infusion.
In other studies, 10 g maltose containing 5 μCi maltose-U-14C were injected into five human subjects and expired CO2 collected for 6 hr. Maximal 14CO2 specific activity was noted at 170 min and a mean of 61.1% of the injected radioactivity was recovered as 14CO2. Less than 8% of the injected 14C was excreted in the urine.
These results indicate that maltose administered intravenously has similar metabolic effects when compared to glucose, and may be efficiently utilized as a carbohydrate substrate. The oxidation of intravenously administered maltose-U14C to 14CO2 demonstrates that circulating maltose is readily metabolized. A solution of maltose could provide twice the mass of sugar (and of calories) per milliliter as an equimolar solution of glucose. Parenterally administered maltose may be of clinical value and should be further studied.
PMCID: PMC292018  PMID: 5552414
15.  Carbohydrate fermentation in the human colon and its relation to acetate concentrations in venous blood. 
Journal of Clinical Investigation  1985;75(5):1448-1454.
There is now substantial evidence that some dietary polysaccharides, notably dietary fiber, escape absorption in the small bowel and are then broken down in the large intestine of man. The main end products of this colonic digestive process, which is anerobic, are short chain fatty acids (SCFA), and acetic, propionic, and butyric acids. Although these acids are known to be absorbed from the colon, their subsequent fate and significance is unknown. We have measured venous blood SCFA levels in healthy subjects after a 16-h fast, and then following oral doses of either 50 mmol SCFA, 5, 10, or 20 g doses of the fermentable carbohydrate lactulose, or 20 g of pectin. Fasting venous blood acetate was 53.8 +/- 4.4 mumol/liter (SEM) (n = 14). Fasting arterial blood acetate, taken simultaneously with venous blood in six subjects, was higher; 125.6 +/- 13.5 mumol/liter (arterial) vs. 61.1 +/- 6.9 mumol/liter (venous). Significant levels of propionate or butyrate were not detected in any blood samples. Following an oral dose of 50 mmol mixed SCFA, venous blood acetate reached a peak of 194.1 +/- 57.9 mumol/liter at 45 min and returned to fasting levels at 2 h. Blood acetate also rose in response to lactulose, peak levels occurring 2-4 h after the dose: 5 g, 98.6 +/- 23.1 mumol/liter; 10 g, 127.3 +/- 18.2 mumol/liter; and 20 g, 181.3 +/- 23.9 mumol/liter. Pectin fermentation was much slower, with blood acetate levels starting to rise after 6 h and remaining elevated at about twice fasting levels for the subsequent 18 h. However, areas under the blood acetate curves were closely related (r = 0.97; n = 5), whatever the source of acetate. These studies show that the large intestine makes an important contribution to blood acetate levels in man and that fermentation may influence metabolic processes well beyond the wall of this organ.
PMCID: PMC425482  PMID: 3998144
16.  Abnormal Carbohydrate Metabolism in Chronic Renal Failure 
To delineate the potential role of disordered glucose and glucose-precursor kinetics in the abnormal carbohydrate metabolism of chronic renal failure, alanine and glucose production and utilization and gluconeogenesis from alanine were studied in patients with chronic compensated renal insufficiency and in normal volunteers. With simultaneous primed injection-continuous infusions of radiolabeled alanine and glucose, rates of metabolite turnover and precursor-product interrelationships were calculated from the plateau portion of the appropriate specific activity curves. All subjects were studied in the postabsorption state. In 13 patients with chronic renal failure (creatinine = 10.7±1.2 mg/100 ml; mean±SEM), glucose turnover was found to be 1,035±99.3 μmol/min. This rate was increased 56% (P = 0.003) over that observed in control subjects (664±33.5 μmol/min). Alanine turnover was 474±96.0 μmol/min in azotemic patients. This rate was 191% greater (P = 0.007) than the rate determined in control subjects (163±19.4 μmol/min). Gluconeogenesis from alanine and the percent of glucose production contributed by gluconeogenesis from alanine were increased in patients with chronic renal failure (192% and 169%, respectively) as compared to controls (P < 0.05 for each). Alanine utilization for gluconeogenesis was increased from 40.2±3.86 μmol/min in control subjects to 143±39.0 μmol/min in azotemic patients (P < 0.05). The percent of alanine utilization accounted for by gluconeogenesis was not altered in chronic renal insufficiency. In nondiabetic azotemic subjects, mean fasting glucose and immunoreactive insulin levels were increased 24.3% (P = 0.005) and 130% (P = 0.046), respectively.
These results in patients with chronic renal failure demonstrate (a) increased glucose production and utilization, (b) increased gluconeogenesis from alanine, (c) increased alanine production and utilization, and (d) a relative impairment to glucose disposal. We conclude that chronic azotemia is characterized by increased rates of glucose and glucose precursor flux and by a relative impairment to glucose disposal. These findings may suggest an underlying hepatic and peripheral insensitivity to the metabolic action of insulin in patients with chronic renal insufficiency.
PMCID: PMC371732  PMID: 659634
17.  Effects of a fibre-enriched milk drink on insulin and glucose levels in healthy subjects 
Nutrition Journal  2009;8:45.
Background
The glycaemic response to foods is dependent on the quality and content of carbohydrates. Carbohydrates in the form of dietary fibre have favourable effects on insulin and glucose metabolism and may help to control energy intake. Dairy products have a relatively low carbohydrate content, and most of the carbohydrate is in the form of lactose which causes gastrointestinal symptoms in part of the population. In order to avoid these symptoms, dairy products can be replaced with lactose-free dairy products which are on the market in many parts of the world. However, the effects of lactose-free products on insulin and glucose metabolism have not been studied.
Methods
In the present study, we investigated the effects of 1) a lactose-free milk drink, 2) a novel fibre-enriched, fat- and lactose-free milk drink and 3) normal fat-free milk on serum glucose and insulin levels and satiety using a randomized block design. Following an overnight fast, 26 healthy volunteers ingested 200 ml of one of these drinks on three non-consecutive days. Insulin and glucose levels and subjective satiety ratings were measured before the ingestion of the milk product and 20, 40, 60, 120 and 180 minutes after ingestion. The responses were calculated as the area under the curve subtracted by the baseline value (AUC minus baseline).
Results
The insulin response was significantly lower for the fibre-enriched milk drink than it was for the other milk products (AUC, P = 0.007). There were no differences in the response for glucose or in the AUC for the subjective satiety ratings between the studied milk products.
Conclusion
The present results suggest that this novel milk drink could have positive effects on insulin response.
doi:10.1186/1475-2891-8-45
PMCID: PMC2760572  PMID: 19796383
18.  Interactions of Fat and Carbohydrate Metabolism—New Aspects and Therapies 
NO one has so far produced anything approaching a clear picture of either fat or carbohydrate metabolism and the interactions of the two are still more involved and elusive although they clearly exist. Plants and animals build up reserves of fat from carbohydrate, but the reverse process (fat into carbohydrate), proved in plant seeds, is still unproven in animals, although theoretically possible.
In normal human metabolism fat-carbohydrate interactions are almost hidden. The disturbances shown in the metabolism of a diabetic seem to give us the clearest indications of these interactions. Either carbohydrate or fat can be used as the main source of body fuel, but their metabolic course is very different, both as regards chemistry and function. It is only whep carbohydrate is not available, either in starvation or severe diabetes, that fat provides the fuel of the body; this contrast is also manifest in the blood and internal organs, especially the liver. Under the commonest normal conditions of diet carbohydrate is predominantly and preferentially used for metabolism. The liver is rich in glycogen, poor in fat; the blood fat is minimal and ketone bodies, although perhaps present in small amount in the blood at most times, are absent on common tests. As soon as carbohydrate is insufficiently available for the needs of metabolism, depot fat flows to the liver and is there catabolized to ketone bodies which recent proof has shown to be burned peripherally in the muscles independent of carbohydrate metabolism. This is a normal process, harmful only in diabetes, and especially harmful when it occurs suddenly, e.g. when insulin is cut off from a fat diabetic dog or human patient. A diabetic supports with ease a prolonged severe ketosis but suffers from one of sudden onset, although of milder severity. Insulin in the diabetic and sugar in the starved switches metabolism from fat to carbohydrate usage very quickly and ketonuria usually disappears in three to six hours.
“Diabetic obesity” is very common and is often seen in the earliest stages and again after insulin treatment. It seems probable that hyperglycæmia causes this obesity and this has been clearly established by observations on an unusual case of lipæmia, diabetes and lipodystrophy.
Lipæcmia may occur in two opposite phases of metabolism, one anabolic—when fat is on its way to storage, the other catabolic—when it is flowing from stores to the liver. The latter is the usual condition obvious in disease.
Work has also been done which suggests that other lipotropic factors—choline, lipocaic, &c., exert an influence on carbohydrate-fat balance, more specifically the glycogen-fat balance in the liver.
In America attention has been drawn to the frequent and persistenzt occurrence of fatty enlargement of the liver in diabetic children. The author has seen many diabetic children (usually in a state of chronic ketosis) with enlarged livers, but such enlargement has rapidly disappeared with better management of the diabetes. Only two out of some 500 diabetic children have clearly shown the unmistakable syndrome of “hepatomegalic dwarfism ”. In these two cases choline and lipocaic were given over prolonged periods without any effect: the liver, however, of one of these cases has since become normal by the addition of zinc protamine insulin.
PMCID: PMC1998199  PMID: 19992415
19.  Effects of intravenously administered fructose and glucose on splanchnic amino acid and carbohydrate metabolism in hypertriglyceridemic men. 
Journal of Clinical Investigation  1975;56(4):970-977.
Splanchnic metabolism was studied in the fed state during prolonged intravenous administration (30 g/h) of either fructose or glucose to hypertriglyceridemic men who had been maintained on a high-carbohydrate diet for 2 wk. Splanchnic exchange of amino acids and carbohydrates was quantified by measurement of splanchnic flow and of blood or plasma arteriohepatic venous concentration gradients. Results obtained in subjects receiving fructose were compared with those obtained in (a) similar subjects receiving glucose and (b) postabsorptive controls maintained on isocaloric, balanced diets. Mean arterial plasma levels of alanine, glycine, serine, threonine, methionine, proline, valine, leucine, histidine, lysine, and ornithine were significantly higher in subjects given fructose than in those give glucose (P less than 0.05). The mean arterial concentration and splanchnic uptake of alanine were significantly higher in subjects given fructose than in postabsorptive controls, despite a significantly lower fractional extraction of alanine in the former (P less than 0.05). The mean arterial plasma levels of serine and ornithine were significantly lower in subjects receiving fructose than in postabsorptive controls (P less than 0.05). About half of the administered fructose or glucose was taken up in the splanchnic region, where approximately 15% was converted to CO2 and 10% to lactate. Half of the fructose taken up in the splanchnic region was converted to glucose released from the liver. The amount of hexose carbon remaining for hepatic synthesis of liquids in subjects given fructose was less than half of that of subjects given glucose. These studies demonstrate that fructose and glucose have divergent effects on amino acid metabolism and that during hypercaloric infusion of glucose (as with fructose), the human liver is a major site of lactate production.
PMCID: PMC301953  PMID: 1159097
20.  Influence of a 60-hour fast on insulin-mediated splanchnic and peripheral glucose metabolism in humans. 
A brief period of starvation (2-3) depletes the hepatic glycogen stores but results in only a limited reduction of the muscle glycogen depots. In this situation insulin resistance contributes to the glucose intolerance, but it is not known which tissue or tissues are responsible for the decreased insulin sensitivity. The present study was therefore undertaken to examine the influence of a 60-h fast on insulin sensitivity in splanchnic and peripheral tissues in normal humans. Euglycemic (95 mg/dl) 1-mU insulin and hyperglycemic (215-225 mg/dl) glucose clamp studies were conducted for 2 h in overnight (12 h) and prolonged (60 h) fasted nonobese subjects. Splanchnic exchange of glucose and gluconeogenic precursors was measured using the hepatic vein catheter technique. During the euglycemic clamp, insulin infusion resulted in similar steady state insulin levels in 60-h and 12-h fasted subjects (73 +/- 7 vs. 74 +/- 5 microU/ml). Total glucose disposal was reduced by 45% after 60 h of fasting (4.0 +/- 0.3 vs. 7.6 +/- 1.1 mg/kg per min, P less than 0.05) and the splanchnic glucose balance reverted from a net release in the basal state (12 h fast, -1.7 +/- 0.2, and 60-h fast, -0.9 +/- 0.1 mg/kg per min, P less than 0.01) to a net uptake during the clamps that was similar after 60 h and 12 h of fasting (0.6 +/- 0.1 vs. 0.6 +/- 0.2 mg/kg per min). During the hyperglycemic clamp, insulin levels rose rapidly in all subjects. In the 12-h fasted group this rise was followed by a further gradual one, reaching significantly higher values than in 60-h fasted subjects during the second hour (67 +/- 15 vs. 25 +/- 2 microU/ml, P less than 0.05). Total glucose disposal was lower, though not significantly so, after the 60-h fast (2.6 +/- 0.4 vs. 5.4 +/- 1.3 mg/kg per min, 0.05 less than P less than 0.10), and as with the euglycemic clamp, the splanchnic glucose balance was altered from a basal net release to a net uptake during the clamp (1.3 +/- 0.2 vs. 1.1 +/- 0.2 mg/kg per min). After an overnight fast, splanchnic lactate uptake fell and the arterial lactate concentration rose in response to both hyperglycemia and hyperinsulinemia, whereas these variables were unchanged in the 60-h fasted subjects during both types of clamp studies.
PMCID: PMC423715  PMID: 3894423
21.  Understanding the physiology of Lactobacillus plantarum at zero growth 
The physiology of Lactobacillus plantarum at extremely low growth rates, through cultivation in retentostats, is much closer to carbon-limited growth than to stationary phase, as evidenced from transcriptomics data, metabolic fluxes, and biomass composition and viability.Using a genome-scale metabolic model and constraint-based computational analyses, amino-acid fluxes—in particular, the rather paradoxical excretion of Asp, Arg, Met, and Ala—could be rationalized as a means to allow extensive metabolism of other amino acids, that is, that of branched-chain and aromatic amino acids.Catabolic products from aromatic amino acids are known to have putative plant-hormone action. The metabolism of amino acids, as well as transcription data, strongly suggested a plant environment-like response in slow-growing L. plantarum, which was confirmed by significant effects of fermented medium on plant root formation.
Natural ecosystems are usually characterized by extremely low and fluctuating nutrient availability. Hence, microorganisms in these environments live a ‘feast-and-famine' existence, with famine the most habitual state. As a result, extremely slow or no growth is the most common state of bacteria, and maintenance processes dominate their life.
In the present study, Lactobacillus plantarum was used as a model microorganism to investigate the physiology of slow growth. Besides fermented foods, this microorganism can be observed in a variety of environmental niches, including plants and lakes, in which nutrient supply is limited. To mimic these conditions, L. plantarum was grown in a glucose-limited chemostat with complete biomass retention (retentostat). During cultivation, biomass progressively accumulated, resulting in steadily decreasing specific substrate availability. Less energy was thus available for growth, and the specific growth rate decreased accordingly, with a final calculated doubling time greater than one year. Detailed measurements of metabolic fluxes were used as constraints in a genome-scale metabolic model to precisely calculate the amount of energy used for net biomass synthesis and for maintenance purposes: at the lowest growth rate investigated (μ=0.0002 h−1), maintenance accounted for 94% of all energy expenses.
Genome-scale metabolic analysis was used in combination with transcriptomics to study the adaptation of L. plantarum to extremely slow growth under limited carbon and energy supply. Importantly, slow growth as investigated here was fundamentally different from the widely studied carbon starvation-induced stationary phase: non-growing cells in retentostat conditions were glucose limited rather than starved, and the transition from a growing to a non-growing state under retentostat conditions was progressive, in contrast with the abrupt transition in batch cultures. These differences were reflected in various aspects of the cell physiology.
The metabolic behavior was remarkably stable during adaptation to slow growth. Although carbon catabolite repression was clearly relieved, as indicated by the upregulation of genes for the utilization of alternative carbohydrates, the metabolism remained largely based on the conversion of glucose to lactate.
Stress resistance mechanisms were also not massively induced. In particular, analysis of the biomass composition—which remained similar to fast-growing cells even under virtually non-growing conditions—and of the gene expression profile, failed to reveal clear stringent or general stress responses, which are generally triggered in glucose-starved cells. The observation that genes involved in growth-associated processes were not downregulated suggested that active synthesis of biomass components (RNA, proteins, and membranes) was required to account for the observed stable biomass and that turnover of macromolecules was high in slow-growing cells. Biomass viability or morphology was also not affected, compared with faster growth conditions. The only typical stress response was the induction of an SOS response—in particular, the upregulation of the two error-prone DNA polymerases—suggesting an increased potential for genetic diversity under adverse conditions. Although diversity was not apparent under the conditions studied here, such mechanisms of increased rates of mutagenesis are likely to have an important role in the adaptation of L. plantarum to slow growth.
A surprising response of L. plantarum during adaptation to slow growth was the production of several amino acids (Arg, Asp, Met, and Ala). A priori, this metabolic behavior seemed inefficient in a context of energy limitation. However, reduced cost analysis using the genome-scale metabolic model indicated that it had a positive effect on energy generation. In-depth analysis of metabolic flux distributions showed that biosynthesis of these amino acids was connected to the catabolism of branched-chain and aromatic amino acids (BCAAs and AAAs), under conditions of limited ammonium efflux. At a fixed ammonium efflux—fixed at the measured value—flux balance analysis indicated that BCAAs and AAAs were expensive to metabolize, because the regeneration of 2-ketoglutarate through glutamate dehydrogenase was limited by ammonium dissipation. Therefore, alternative pathways had to be active to supply the necessary pool of 2-ketoglutarate. At low growth rates, amino-acid production (Arg, Asp, Ala, and Met) accounted for most of the 2-ketoglutarate regeneration. Although it came at the expense of ATP, this metabolic alternative to glutamate dehydrogenase was less energy costly than other solutions such as purine biosynthesis. This is thus an excellent example in which precise, quantitative modeling results in new insights in physiology that intuition would never have achieved. It also shows that flux balance analysis can be used to accurately predict energetically inefficient metabolism, provided the appropriate fluxes are constrained (here, ammonium efflux).
The observation that BCAAs and AAAs were catabolized at the expense of energy was intriguing. However, several end products of these catabolic pathways can serve as signaling molecules for interactions with other organisms. In particular, precursors of plant hormones were predicted as possible end products in the model simulations. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. The metabolic analysis thus suggested that slow-growing L. plantarum produced plant hormones—or precursors thereof—as a strategy to divert the plant metabolism towards its own interest. In support of this view, transcriptome analysis indicated the upregulation of genes involved in the catabolism of β-glucosides—typical sugars from plant cell wall—as well as a very high induction of six gene clusters encoding cell-surface protein complexes predicted to have a role in the utilization of plant polysaccharides (csc clusters). In such a plant context, limited ammonium production would also make sense, because of the well-documented toxicity of ammonium for plants: production of amino acids could represent an alternative to ammonium excretion while keeping both parties satisfied.
In conclusion, the physiology of L. plantarum at extremely low growth rates, as studied by genome-scale metabolic modeling and transcriptomics, is fundamentally different from that of starvation-induced stationary phase cells. Excitingly, these conditions seem to trigger responses that favor interactions with the environment, more specifically with plants. The reported observations were made in the absence of any plant-derived material, suggesting that this response might constitute a hardwired behavior.
Situations of extremely low substrate availability, resulting in slow growth, are common in natural environments. To mimic these conditions, Lactobacillus plantarum was grown in a carbon-limited retentostat with complete biomass retention. The physiology of extremely slow-growing L. plantarum—as studied by genome-scale modeling and transcriptomics—was fundamentally different from that of stationary-phase cells. Stress resistance mechanisms were not massively induced during transition to extremely slow growth. The energy-generating metabolism was remarkably stable and remained largely based on the conversion of glucose to lactate. The combination of metabolic and transcriptomic analyses revealed behaviors involved in interactions with the environment, more particularly with plants: production of plant hormones or precursors thereof, and preparedness for the utilization of plant-derived substrates. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. Thus, conditions of slow growth and limited substrate availability seem to trigger a plant environment-like response, even in the absence of plant-derived material, suggesting that this might constitute an intrinsic behavior in L. plantarum.
doi:10.1038/msb.2010.67
PMCID: PMC2964122  PMID: 20865006
Lactobacillus plantarum; metabolic modeling; retentostat; slow growth; transcriptome analysis
22.  Effects of a late supper on digestion and the absorption of dietary carbohydrates in the following morning 
Background
Our previous experiment showed that the light intensity exposed on the subjects during evening time had no effect in the following morning on the efficiency of the digestion and absorption of dietary carbohydrates ingested at a usual suppertime. People who keep late hours usually have a late suppertime; thus, we examined the effects of a late suppertime on gastrointestinal activity in the following morning in comparison to that of a usual suppertime.
Methods
Twelve female university students volunteered as paid participants. The breath hydrogen test was carried out to estimate the amount of unabsorbed dietary carbohydrates and the percentage of the total amount of dietary carbohydrates in the breakfast that were unabsorbed, as well as to estimate oro-cecal transit time. The respiratory quotient was also measured to find the ratio of carbohydrates/lipid metabolism in the post-breakfast state. Subjects’ peripheral blood glucose concentration was measured by a blood glucose meter. The subjects participated under two different experimental conditions: with a usual suppertime (having supper at 18:00) and a late suppertime (having supper at 23:00).
Results
The efficiency of the digestion and absorption of dietary carbohydrates in the breakfast under late suppertime conditions was higher than that under usual suppertime conditions. Usual or late suppertime had no effect on the ratio of carbohydrates to lipids oxidized after the subjects had breakfast. There were significant differences in the blood glucose level between the two conditions at 30, 60, 120, 150, and 180 minutes after having breakfast, whereas the mean blood glucose level under late suppertime conditions was significantly higher than under usual suppertime conditions.
Conclusions
Having a late supper showed a worse effect on postprandial serum glucose profiles the following morning. This study confirmed that keeping our usual meal timing is important for our health.
doi:10.1186/1880-6805-32-9
PMCID: PMC3685573  PMID: 23705984
Suppertime; Carbohydrate absorption; Postprandial serum glucose profile; Breath hydrogen test
23.  Temporal Changes in Plasma Concentration of Leptin, IGF-1, Insulin and Metabolites Under Extended Fasting and Re-Feeding Conditions in Growing Lambs 
Background
A fall in plasma concentration of energy status related hormones (leptin, insulin-like growth factor-1 (IGF-1) and insulin) and body energy expenditure occurs in response to short term fasting. Nevertheless, the relations of the fasting-induced changes in energy related hormones and metabolites with fasting energy expenditure (FEE) under extended fasting condition have received little attention so far.
Objectives
It is not clear how energy status related hormones coordinate to cope with feed deprivation under extended fasting time conditions and how quickly these hormones re-bound to fed-state values in response to re-feeding. Thus the objectives of this study were: 1) to determine the effects of extended fasting on plasma concentration of leptin, IGF-1, insulin, glucose, NEFA, 3-β-hydroxybutyrate (BOHB) and urea; and 2) to study the relations of energy status related hormones with FEE and substrate oxidations under extended fasting conditions.
Materials and Methods
Eighteen six-month-old growing lambs (9 females and 9 males) were fasted for three days. Blood samples were taken one hour before (-1H) and 48 and 72 hours after fasting (48H and 72H) and two hours after re-feeding (+2H) from jugular vein. During the last 22 hours of fasting, gas exchange (CO2 production and O2 consumption) were measured using an open-circuit indirect calorimeter. Respiratory quotient (RQ), FEE and relative proportions of oxidized protein, fat and carbohydrate were calculated.
Results
Plasma levels of leptin, insulin, IGF-1 and glucose decreased but NEFA and urea levels increased within 48H of fasting. Concentration of insulin significantly increased with extended fasting while leptin and IGF-1 levels remained constant. Glucose was the only blood variable that showed a quick re-bound within two hours after re-feeding. Leptin and IGF-1 showed significant positive relations with glucose and BOHB but negative relations with NEFA and Urea. Carbohydrate, fat and proteins contributed to 17%, 61% and 22% of FEE respectively in three-day-fasted lambs. FEE was negatively correlated with insulin and NEFA concentrations in plasma.
Conclusions
Even though plasma levels of leptin and IGF-1 decreased and remained constant under extended fasting, neither leptin nor IGF1 re-bounded to fed-status values within two hours after re-feeding. Under extended fasting condition, firstly an insulin resistance develops and secondly, a fall in FEE through a switch from carbohydrate- to fat-based metabolism occurs and there is an evident negative correlation between FEE and plasma concentration of NEFA.
doi:10.5812/ijem.6529
PMCID: PMC3693659  PMID: 23853618
Leptin; Insulin-Like Growth Factor I; Energy Metabolism; Sheep
24.  Control of ATP homeostasis during the respiro-fermentative transition in yeast 
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content. Transient accumulation of the purine salvage pathway intermediate inosine accounts for the apparent loss of adenine nucleotides.Inosine formation in response to perturbations of cellular energy balance depends on the presence of a fermentable carbon source. Under respiratory conditions, AMP accumulates instead and no inosine is formed.Conversion of AXPs into inosine is facilitated by AMP deaminase, Amd1, and IMP-specific 5'-nucleotidase, Isn1. Inosine recycling into the AXP pool is facilitated by the purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1.Impaired inosine formation results in altered metabolite pool dynamics in response to glucose addition, but does not change glycolytic flux. However, mutants blocked in inosine formation exhibit delayed growth acceleration after glucose addition.
Yeast cells are exposed to strongly fluctuating nutrient concentrations in their natural environment, which requires rapid and efficient adaptation through rearrangements on all levels of their metabolism. The quantitative understanding of these adaptation processes represents the basis for a directed optimization of the microorganism to suit the needs of biochemical production processes that often impose non-uniform or harsh cultivation conditions (Lara et al, 2006), or require a redirection of metabolic fluxes to improve productivity (Bailey, 1991). In this context, controlled perturbation experiments represent a valuable tool, as they allow studying the transition from one defined physiological state to another under well-characterized conditions. Measurements of metabolite pool dynamics and enzymatic activities in response to different perturbations enable the quantitative mathematical analysis of glycolytic dynamics, which is ultimately meant to discriminate the impact of allosteric regulation and changes in the enzymatic make-up of the cell on the overall metabolic response (Rizzi et al, 1997; Teusink et al, 2000; Daran-Lapujade et al, 2007; van den Brink et al, 2008).
A long-standing problem in the context of these studies was the apparent loss of adenine nucleotides, which immediately follows the relief from glucose limitation (Theobald et al, 1997; Kresnowati et al, 2006). In this study, we showed that the transient accumulation of the purine salvage pathway (PSP) metabolites, IMP and inosine, account for the loss of AXP nucleotides. The pathway for inosine formation and recycling was identified, and the interplay between different pathways during the respiro-fermentative transition is schematically summarized in Figure 10. The presence of a quickly metabolizable sugar causes accumulation of the phosphorylated metabolites F1,6P, T6P and G3P, which results in a transient imbalance of ATP-consuming and ATP-regenerating reactions, and provokes a drop in both ATP and intracellular phosphate (Pi) concentrations. The joint action of fast ATP consumption and the Adk1 reaction results in the net-production of AMP. The accumulation of AMP, however, is prevented by Amd1 and Isn1, which readily convert AMP via IMP into inosine. Recycling of inosine into IMP is facilitated by Pnp1 and the concomitant action of Hpt1 and Xpt1, with Hpt1 having the predominant role. We suggest that inosine formation serves to prevent unscheduled AMP accumulation, and to store AXP nucleotides in a metabolically ‘neutral' form until re-equilibration of glycolysis eventually allows recovery of ATP levels. Recycling of inosine via IMP represents an energy-saving way to replenish the AXP pool, as de novo synthesis of IMP starting from PRPP requires 4 ATP molecules, whereas IMP production from inosine and PRPP is an ATP-neutral process (Figure 10).
In contrast to the behavior observed in the presence of fermentable carbon sources, neither IMP nor inosine were formed in response to perturbations of the cellular energy balance under respiratory conditions. Instead, the drop in ATP level caused a concomitant increase in AMP concentration. Therefore, we conclude that the conversion of AXP nucleotides into inosine represents a specific short-term metabolic response to the perturbation of cellular energy homeostasis, which is controlled by the presence of a fermentable carbon source (or a sugar derivative that can undergo rapid phosphorylation). The discrimination between AMP accumulation and AMP-to-inosine conversion seems to be controlled by Amd1 activity. At the present stage of investigation, we cannot conclude whether regulation of Amd1 is brought about by allosteric control or by posttranslational modification. However, given that inorganic phosphate is a potent inhibitor of Amd1 (Merkler et al, 1989) and intracellular phosphate concentration transiently drops when cells become exposed to fermentable carbon sources (our data, and e.g. Hohmann et al, 1996), phosphate availability is likely to have a pivotal role in the regulation of AXP pool size and inosine formation.
The impact of defective AXP cycling on the global metabolic response to glucose addition was tested under conditions in which respiration was inhibited by antimycin A. The amd1 mutant, in which adenine nucleotide cycling was completely blocked, showed the strongest deviations from the wild-type behavior. In this mutant, adenylic energy charge exhibited a strong drop after glucose addition, and recovered much slower than in wild-type cells. Furthermore, deletion of AMD1 resulted in strong accumulation of AMP and pronounced changes in the dynamics of trehalose-6-phosphate, glycerol-3-phosphate, and PRPP after glucose addition (Figure 6).
Despite pronounced changes in metabolite pool dynamics caused by the deletion of AMD1, no alterations in the production of the fermentative end products, ethanol and glycerol, nor in the consumption of glucose were observed (Figure 9B). However, the amd1 and isn1 mutant strains showed delayed growth acceleration after glucose addition (Figure 9C). The observation of unaltered fermentation capacity but concomitant delay in growth acceleration in the amd and isn1 mutants points to the possibility that regulation of glycolysis is not the major target of AMP. Indeed, our study revealed a delayed increase of PRPP concentration in the amd1 strain after glucose addition. The PRPP protein has a central role as a precursor for purine nucleotide de novo synthesis and the synthesis of amino acids (Vavassori et al, 2005). Hence, limitation of this important precursor may negatively affect growth. In addition, AMP accumulation may have an important signaling function acting, for example, through the cAMP/PKA pathway (Thevelein et al, 2005; Zaman et al, 2009) and/or via Snf1 (Celenza and Carlson, 1984). However, experimental evidence to support this potential link is missing. Thus, the assumption of a signaling role of AMP remains speculative, although intriguing, when asking for the actual function of AXP-to-inosine conversion during the respiro-fermentative transition.
On the basis of this study, we put forward the implication of adenine nucleotide cycling through the PSP in energy homeostasis in yeast. Answering the question whether this pathway is also active in humans could potentially contribute to a better understanding of metabolic processes that control the metabolic transition from respiratory to respiro-fermentative energy supply in muscle upon heavy exercise. In particular, it may help to understand conflicting results on the impaired physical performance of individuals carrying an AMP deaminase dysfunction under these conditions (Tarnopolsky et al, 2001; Fischer et al, 2007).
Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content ([ATP]+[ADP]+[AMP]=[AXP]). The unknown fate of ‘lost' AXP nucleotides represented a long-standing problem for the understanding of the yeast's physiological response to changing growth conditions. Transient accumulation of the purine salvage pathway intermediate, inosine, accounted for the apparent loss of adenine nucleotides. Conversion of AXPs into inosine was facilitated by AMP deaminase, Amd1, and IMP-specific 5′-nucleotidase, Isn1. Inosine recycling into the AXP pool was facilitated by purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1. Analysis of changes in 24 intracellular metabolite pools during the respiro-fermentative growth transition in wild-type, amd1, isn1, and pnp1 strains revealed that only the amd1 mutant exhibited significant deviations from the wild-type behavior. Moreover, mutants that were blocked in inosine production exhibited delayed growth acceleration after glucose addition. It is proposed that interconversion of adenine nucleotides and inosine facilitates rapid and energy-cost efficient adaptation of the AXP pool size to changing environmental conditions.
doi:10.1038/msb.2009.100
PMCID: PMC2824524  PMID: 20087341
ATP homeostasis; metabolic regulation; purine nucleotide metabolism; respiro-fermentative transition; yeast
25.  Mitochondrial 2,4-dienoyl-CoA Reductase Deficiency in Mice Results in Severe Hypoglycemia with Stress Intolerance and Unimpaired Ketogenesis 
PLoS Genetics  2009;5(7):e1000543.
The mitochondrial β-oxidation system is one of the central metabolic pathways of energy metabolism in mammals. Enzyme defects in this pathway cause fatty acid oxidation disorders. To elucidate the role of 2,4-dienoyl-CoA reductase (DECR) as an auxiliary enzyme in the mitochondrial β-oxidation of unsaturated fatty acids, we created a DECR–deficient mouse line. In Decr−/− mice, the mitochondrial β-oxidation of unsaturated fatty acids with double bonds is expected to halt at the level of trans-2, cis/trans-4-dienoyl-CoA intermediates. In line with this expectation, fasted Decr−/− mice displayed increased serum acylcarnitines, especially decadienoylcarnitine, a product of the incomplete oxidation of linoleic acid (C18:2), urinary excretion of unsaturated dicarboxylic acids, and hepatic steatosis, wherein unsaturated fatty acids accumulate in liver triacylglycerols. Metabolically challenged Decr−/− mice turned on ketogenesis, but unexpectedly developed hypoglycemia. Induced expression of peroxisomal β-oxidation and microsomal ω-oxidation enzymes reflect the increased lipid load, whereas reduced mRNA levels of PGC-1α and CREB, as well as enzymes in the gluconeogenetic pathway, can contribute to stress-induced hypoglycemia. Furthermore, the thermogenic response was perturbed, as demonstrated by intolerance to acute cold exposure. This study highlights the necessity of DECR and the breakdown of unsaturated fatty acids in the transition of intermediary metabolism from the fed to the fasted state.
Author Summary
Fatty acids released from triacylglycerols or obtained from the diet serve as a main energy provider to the heart and skeletal muscle, and when carbohydrates are scarce, fatty acids provide energy for the whole organism. Inherited disorders of mitochondrial β-oxidation are among the most common inborn errors of metabolism affecting infants and children. Under normal conditions, patients are usually asymptomatic; but when challenged with metabolic stress, severe phenotypes arise. Here we describe the generation of a mouse model in which the total degradation of unsaturated fatty acids is prevented by disruption of an auxiliary enzyme of β-oxidation. Although degradation of saturated fatty acids proceeds normally, the phenotype presented here is in many ways similar to mouse models of the disrupted classical β-oxidation pathway, but with additional unique features. The null mutant mice are asymptomatic until exposed to fasting, during which they switch on ketogenesis, but simultaneously develop hypoglycemia. A number of human patients suffer from idiopathic hypoglycemia (hypoglycemia of unknown cause). Our mouse model links this disease state to a specific defect in the breakdown of polyunsaturated fatty acids. Furthermore, it shows that degradation of unsaturated fatty acids is essential for balanced fatty acid and energy metabolism, as well as adaptation to metabolic stress.
doi:10.1371/journal.pgen.1000543
PMCID: PMC2697383  PMID: 19578400

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