Correlation of egg counts of Clonorchis sinensis by three methods of fecal examination

The Kato-Katz (KK) method is a well-known method of fecal examination for helminthiases. Its diagnostic sensitivity was found very high for clonorchiasis. The present study evaluated the correlation of Clonorchis sinensis egg counts by the KK method with those by direct smear and formalin-ether (FE) technique. The egg counts obtained by the KK method (Y) were correlated with the counts by direct smear (X) with the equation of Y = 659.4 + 0.266X (r2 = 0.738), but not with those by the FE method. The present study demonstrated that the KK method and direct smear were useful for both qualitative and quantitative diagnosis of clonorchiasis, especially in the field.     

Real-time PCR Demonstrates High Prevalence of Schistosoma japonicum in the Philippines: Implications for Surveillance and Control

Background

The Philippines has a population of approximately 103 million people, of which 6.7 million live in schistosomiasis-endemic areas with 1.8 million people being at risk of infection with Schistosoma japonicum. Although the country-wide prevalence of schistosomiasis japonica in the Philippines is relatively low, the prevalence of schistosomiasis can be high, approaching 65% in some endemic areas. Of the currently available microscopy-based diagnostic techniques for detecting schistosome infections in the Philippines and elsewhere, most exhibit varying diagnostic performances, with the Kato-Katz (KK) method having particularly poor sensitivity for detecting low intensity infections. This suggests that the actual prevalence of schistosomiasis japonica may be much higher than previous reports have indicated.

Methodology/Principal Findings

Six barangay (villages) were selected to determine the prevalence of S. japonicum in humans in the municipality of Palapag, Northern Samar. Fecal samples were collected from 560 humans and examined by the KK method and a validated real-time PCR (qPCR) assay. A high S. japonicum prevalence (90.2%) was revealed using qPCR whereas the KK method indicated a lower prevalence (22.9%). The geometric mean eggs per gram (GMEPG) determined by the qPCR was 36.5 and 11.5 by the KK. These results, particularly those obtained by the qPCR, indicate that the prevalence of schistosomiasis in this region of the Philippines is much higher than historically reported.

Conclusions/Significance

Despite being more expensive, qPCR can complement the KK procedure, particularly for surveillance and monitoring of areas where extensive schistosomiasis control has led to low prevalence and intensity infections and where schistosomiasis elimination is on the horizon, as for example in southern China.     

The Right Tool for the Job: Detection of Soil-Transmitted Helminths in Areas Co-endemic for Other Helminths

Background

Due to the recent increased use of the McMaster (MM) fecal egg counting method for assessing benzimidazole drug efficacy for treating soil-transmitted helminth (STH) infections, the aim of the current study was to determine the operational value of including the MM method alongside the Kato-Katz (KK) fecal thick smear to increase the diagnostic sensitivity when STHs are co-endemic with trematode helminths (e.g., Schistosoma mansoni).

Methods

A cross-sectional study was conducted in school-aged children aged 4-18 years in the northeastern region of the State of Minas Gerais (Brazil), where Necator americanus, Ascaris lumbricoides, Trichuris trichiura, and S. mansoni are co-endemic. One fecal sample from each participant was collected and transported to the field laboratory for analysis. Coprological diagnosis was performed on each fecal sample by three different methods: Formalin-Ether Sedimentation (FES), KK and the MM technique. The diagnostic sensitivity and negative predictive value (NPV) of each technique was calculated using the combination of all three techniques as the composite standard. In order to determine the agreement between the three techniques Fleiss´ kappa was used. Both the Cure Rate (CR) and the Fecal Egg Count Reduction (FECR) were calculated using the two quantification techniques (i.e., the MM and KK).

Results

Fecal samples from 1260 children were analyzed. The KK had higher diagnostic sensitivity than the MM for the detection of both A. lumbricoides (KK 97.3%, MM 69.5%) and hookworm (KK 95.1%, MM 80.8%). The CR of a single dose of mebendazole varied significantly between the KK and MM for both A. lumbricoides (p = 0.016) and hookworm (p = 0.000), with lower rates obtained with the KK. On the other hand, the FECR was very similar between both techniques for both A. lumbricoides and hookworm.

Conclusion

The MM did not add any diagnostic value over the KK in areas where both STHs and trematodes were co-endemic. The lower sensitivity of the MM would have an important impact on the administration of selective school-based treatment in this area since if only the MM were used, 36 (13.9%) children diagnosed with A. lumbricoides would have gone untreated.     

Simple Fecal Flotation Is a Superior Alternative to Guadruple Kato Katz Smear Examination for the Detection of Hookworm Eggs in Human Stool

Background

Microscopy-based identification of eggs in stool offers simple, reliable and economical options for assessing the prevalence and intensity of hookworm infections, and for monitoring the success of helminth control programs. This study was conducted to evaluate and compare the diagnostic parameters of the Kato-Katz (KK) and simple sodium nitrate flotation technique (SNF) in terms of detection and quantification of hookworm eggs, with PCR as an additional reference test in stool, collected as part of a baseline cross-sectional study in Cambodia.

Methods/Principle Findings

Fecal samples collected from 205 people in Dong village, Rovieng district, Preah Vihear province, Cambodia were subjected to KK, SNF and PCR for the detection (and in case of microscopy-based methods, quantification) of hookworm eggs in stool. The prevalence of hookworm detected using a combination of three techniques (gold standard) was 61.0%. PCR displayed a highest sensitivity for hookworm detection (92.0%) followed by SNF (44.0%) and quadruple KK smears (36.0%) compared to the gold standard. The overall eggs per gram feces from SNF tended to be higher than for quadruple KK and the SNF proved superior for detecting low egg burdens.

Conclusion/Significance

As a reference, PCR demonstrated the higher sensitivity compared to SNF and the quadruple KK method for detection of hookworm in human stool. For microscopic-based quantification, a single SNF proved superior to the quadruple KK for the detection of hookworm eggs in stool, in particular for low egg burdens. In addition, the SNF is cost-effective and easily accessible in resource poor countries.     

A comparative study of the TF-Test®, Kato-Katz, Hoffman-Pons-Janer, Willis and Baermann-Moraes coprologic methods for the detection of human parasitosis

This study compares the diagnostic accuracy of the TF-Test(?) (TFT) for human parasitosis with results obtained using the traditional Kato-Katz (KK), Hoffman-Pons-Janer (HPJ), Willis and Baermann-Moraes (BM) techniques. Overall, four stool samples were taken from each individual; three alternate-day TFT stool samples and another sample that was collected in a universal container. Stool samples were taken from 331 inhabitants of the community of Quilombola Santa Cruz. The gold standard (GS) for protozoa detection was defined as the combined results for TFT, HPJ and Willis coproscopic techniques; for helminth detection, GS was defined as the combined results for all five coproscopic techniques (TFT, KK, HPJ, Willis and BM). The positivity rate of each method was compared using the McNemar test. While the TFT exhibited similar positivity rates to the GS for Entamoeba histolytica/dispar (82.4%) and Giardia duodenalis (90%), HPJ and Willis techniques exhibited significantly lower positivity rates for these protozoa. All tests exhibited significantly lower positivity rates compared with GS for the diagnosis of helminths. The KK technique had the highest positivity rate for diagnosing Schistosoma mansoni (74.6%), while the TFT had the highest positivity rates for Ascaris lumbricoides (58.1%) and hookworm (75%); HPJ technique had the highest positivity rate for Strongyloides stercoralis (50%). Although a combination of tests is the most accurate method for the diagnosis of enteral parasites, the TFT reliably estimates the prevalence of protozoa and selected helminths, such as A. lumbricoides and hookworm. Further studies are needed to evaluate the detection accuracy of the TFT in samples with varying numbers of parasites.     

Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear,faecal flotation and quantitative real-time PCR in human stool

BackgroundMonitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies.Methodology/Principal findingsThe diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO₃) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO₃ flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR (p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30).Conclusions/SignificanceThis study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.     

Evaluation of parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni in a low transmission area

This study evaluated parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni. A population-based study was performed in 201 inhabitants from a low transmission locality named Pedra Preta, municipality of Montes Claros, state of Minas Gerais, Brazil. Four stool samples were analysed using two techniques, the Kato-Katz^® (KK) technique (18 slides) and the TF-Test^®, to establish the infection rate. The positivity rate of 18 KK slides of four stool samples was 28.9% (58/201) and the combined parasitological techniques (KK+TF-Test^®) produced a 35.8% positivity rate (72/201). Furthermore, a polymerase chain reaction (PCR)-ELISA assay produced a positivity rate of 23.4% (47/201) using the first sample. All 72 patients with positive parasitological exams were treated with a single dose of Praziquantel^® and these patients were followed-up 30, 90 and 180 days after treatment to establish the cure rate. Cure rates obtained by the analysis of 12 KK slides were 100%, 100% and 98.4% at 30, 90 and 180 days after treatment, respectively. PCR-ELISA revealed cure rates of 98.5%, 95.5% and 96.5%, respectively. The diagnostic and assessment of cure for schistosomiasis may require an increased number of KK slides or a test with higher sensitivity, such as PCR-ELISA, in situations of very low parasite load, such as after therapeutic interventions.     

Intestinal schistosomiasis: Can a urine sample decide the infection?

Intestinal schistosomiasis, one of the neglected tropical diseases whose control depends on accurate diagnosis of the disease prevalence. The use of low sensitive Kato Katz (KK) fecal egg detection method as a reference gold standard is not an accurate indication especially in low transmission areas. Latent class analysis frameworks especially the Bayesian could be used instead to compare between different diagnostic tests without the use of a gold standard method as a reference. Thus, this study compared two urine-based tests for the detection of circulating antigen and cell free DNA of Schistosoma mansoni versus KK method using the Bayesian latent class analytical framework and in two models where the trace results of point of contact - assay of circulating cathodic antigen (POC-CCA) were once estimated as positive, and as negative in the other model. The Bayesian framework in the trace CCA positive model showed an estimate of disease prevalence of 26% (95% BCI:0 to 60%). POC-CCA showed the highest sensitivity (74% with BCI: 9 to 91%) and lowest specificity for (20% with BCI: 0% to 37%) and the reverse for KK. For POC-CCA with traces considered negative, it was found that results between the three tests were moderated where the positivity for infection by Schistosoma antigen detection and PCR for cell free DNA approached that estimated by the Bayesian framework (44%), and the specificity for point of contact assay(81%; 95%BCI: 59% to 100%) rose in hand with its sensitivity(77%, 95% BCI:53% to 100%) and with results for PCR test (sensitivity = 80%; 95% BCI: 61% to 100%, specificity = 69%; 95% BIC: 47% to 100%). KK remains with the highest specificity while its sensitivity in the two models never exceeded 22%. Thus, we conclude that the use of a single urine sample could be very sensitive and highly specific in the diagnosis of intestinal schistosomiasis using either the trace negative model of point of contact assay, or conventional PCR, when compared to the fecal egg detection using duplicate KK. However, the use of a single tool restricts the management of the disease in areas of low endemicity.     

A Combination of the Kato-Katz Methods and ELISA to Improve the Diagnosis of Clonorchiasis in an Endemic Area,China

Background

Examination of feces by light microscopy is widely used for specific parasitological diagnosis of clonorchiasis. However, the true incidence of infection is underestimated owing to the high missing diagnosis rate of this method. The enzyme-linked immunosorbent assay (ELISA) is widely used for the detection and control of clonorchiasis but the practicality of this method is unclear. The purpose of this study was to evaluate the effect of ELISA as a supplementary method for the diagnosis of clonorchiasis.

Methodology/Principal Findings

The present study recruited 2,359 clinically suspected patients from Heilongjiang Province, China. In all, 954 cases were identified as antibody-positive by immunoglobulin (IgG)-ELISA and 495 individuals were diagnosed as egg-positive by the Kato-Katz (KK) method. The seropositive and egg-negative individuals were re-examined by repeated egg counts and/or the number of KK slides and 18 (18.18%) cases were confirmed as clonorchiasis. The 40.44%, antibody-positive rate determined by IgG-ELISA was significantly higher (P<0.05) than the 21.75% egg-positive rate found by examination of feces. A Bayesian approach indicated that the prevalence of clonorchiasis in this region was 22.27% and that the sensitivity, specificity, positive predictive value and negative predictive value of IgG-ELISA were 98.7%, 76.53%, 54.66% and 99.52%, respectively. The agreement between the two methods was moderate (kappa value = 0.564). The clonorchiasis patients lived mainly along the Songhua River. The risk factors, except for ethnic factors, were estimated effectively by both methods.

Conclusions/Significance

The present study suggested that clonorchiasis was widely distributed in Heilongjiang Province, China. The missing diagnosis rate was high using the KK technique alone. The combination of immunological methods and parasitological techniques could improve diagnostic accuracy and reduce the missing diagnosis rate. ELISA used as an auxiliary diagnostic method was realistic and practical for a large-scale screening test, monitoring the prevalence and assessing the risk factors of clonorchiasis.     

Affordable artificial intelligence-based digital pathology for neglected tropical diseases: A proof-of-concept for the detection of soil-transmitted helminths and Schistosoma mansoni eggs in Kato-Katz stool thick smears

BackgroundWith the World Health Organization’s (WHO) publication of the 2021–2030 neglected tropical diseases (NTDs) roadmap, the current gap in global diagnostics became painfully apparent. Improving existing diagnostic standards with state-of-the-art technology and artificial intelligence has the potential to close this gap.Methodology/Principal findingsWe prototyped an artificial intelligence-based digital pathology (AI-DP) device to explore automated scanning and detection of helminth eggs in stool prepared with the Kato-Katz (KK) technique, the current diagnostic standard for diagnosing soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura and hookworms) and Schistosoma mansoni (SCH) infections. First, we embedded a prototype whole slide imaging scanner into field studies in Cambodia, Ethiopia, Kenya and Tanzania. With the scanner, over 300 KK stool thick smears were scanned, resulting in total of 7,780 field-of-view (FOV) images containing 16,990 annotated helminth eggs (Ascaris: 8,600; Trichuris: 4,083; hookworms: 3,623; SCH: 684). Around 90% of the annotated eggs were used to train a deep learning-based object detection model. From an unseen test set of 752 FOV images containing 1,671 manually verified STH and SCH eggs (the remaining 10% of annotated eggs), our trained object detection model extracted and classified helminth eggs from co-infected FOV images in KK stool thick smears, achieving a weighted average precision (± standard deviation) of 94.9% ± 0.8% and a weighted average recall of 96.1% ± 2.1% across all four helminth egg species.Conclusions/SignificanceWe present a proof-of-concept for an AI-DP device for automated scanning and detection of helminth eggs in KK stool thick smears. We identified obstacles that need to be addressed before the diagnostic performance can be evaluated against the target product profiles for both STH and SCH. Given that these obstacles are primarily associated with the required hardware and scanning methodology, opposed to the feasibility of AI-based results, we are hopeful that this research can support the 2030 NTDs road map and eventually other poverty-related diseases for which microscopy is the diagnostic standard.     

A Latent Markov Modelling Approach to the Evaluation of Circulating Cathodic Antigen Strips for Schistosomiasis Diagnosis Pre- and Post-Praziquantel Treatment in Uganda

Regular treatment with praziquantel (PZQ) is the strategy for human schistosomiasis control aiming to prevent morbidity in later life. With the recent resolution on schistosomiasis elimination by the 65th World Health Assembly, appropriate diagnostic tools to inform interventions are keys to their success. We present a discrete Markov chains modelling framework that deals with the longitudinal study design and the measurement error in the diagnostic methods under study. A longitudinal detailed dataset from Uganda, in which one or two doses of PZQ treatment were provided, was analyzed through Latent Markov Models (LMMs). The aim was to evaluate the diagnostic accuracy of Circulating Cathodic Antigen (CCA) and of double Kato-Katz (KK) faecal slides over three consecutive days for Schistosoma mansoni infection simultaneously by age group at baseline and at two follow-up times post treatment. Diagnostic test sensitivities and specificities and the true underlying infection prevalence over time as well as the probabilities of transitions between infected and uninfected states are provided. The estimated transition probability matrices provide parsimonious yet important insights into the re-infection and cure rates in the two age groups. We show that the CCA diagnostic performance remained constant after PZQ treatment and that this test was overall more sensitive but less specific than single-day double KK for the diagnosis of S. mansoni infection. The probability of clearing infection from baseline to 9 weeks was higher among those who received two PZQ doses compared to one PZQ dose for both age groups, with much higher re-infection rates among children compared to adolescents and adults. We recommend LMMs as a useful methodology for monitoring and evaluation and treatment decision research as well as CCA for mapping surveys of S. mansoni infection, although additional diagnostic tools should be incorporated in schistosomiasis elimination programs.     

Evaluation of prekallikrein activator (PKA) activity in plasma-derived preparations

The study was carried out on 111 series of human immunoglobulin preparations (81 series for intravenous use, 30 series for intramuscular use) produced abroad and in Poland. Kallikrein (KK) and prekallikrein activator (PKA) activities were identified using chromogenic substrate. S-2302 and proteinase inhibitors were tested by double immunodiffusion method. Several biochemical, biological and serological methods were also applied which are valid in the control of intravenous and intramuscular immunoglobulin preparations. It was found that trace amounts of PKA and KK are present in human immunoglobulin preparations for intravenous use such as Bioglobulina and Gamma-Venina (PKA 0.0092-0.0505 mu kat/L, and KK 0,-0.0093 mu kat/L). On the other hand normal human immunoglobulin preparations for intramuscular use had somewhat higher level of KK and PKA and anti-HBs Immunoglobulin preparations (5.50 mu kat/L and 153.91 mu kat/L) and Tetabulina preparations had very high levels of these factors as compared to remaining preparations (4.50 mu kat/L and 316.5 mu kat/L).     

Preparation of an organic solvent-tolerant strain from baker's yeast

By using immobilized baker's yeast repeatedly in isooctane with occasional reactivation by cultivation, we succeeded in the preparation of an organic solvent-tolerant strain, named KK21, which could grow in the presence of isooctane. This is the first report on an organic solvent-tolerant strain from baker's yeast. Strain KK21 showed high tolerance to organic solvents and maintained a high and stable activity on continuous reduction of n-butyl 3-oxobutanoate in an isooctane-medium two-phase system. Although the morphology of strain KK21 was the same as that of baker's yeast, the saturated fatty acid occupancy (SFA occupancy), which is defined as the percentage of saturated fatty acids in the total fatty acids of phospholipids, of strain KK21 was significantly higher than that of parental baker's yeast when strain KK21 was grown in the presence of isooctane, suggesting that a decrease in fluidity of the cell membrane might play an important role in the tolerance to organic solvents.     

Comparison of the Kato-Katz and Helmintex methods for the diagnosis of schistosomiasis in a low-intensity transmission focus in Bandeirantes, Paraná, southern Brazil

The diagnosis of schistosomiasis is problematic in low-intensity transmission areas because parasitological methods lack sensitivity and molecular methods are neither widely available nor extensively validated. Helmintex is a method for isolating eggs from large faecal samples. We report preliminary results of a comparative evaluation of the Helmintex and Kato-Katz (KK) methods for the diagnosis of schistosomiasis in a low-intensity transmission area in Bandeirantes, Paraná, southern Brazil. Eggs were detected by both methods in seven patients, whereas only Helmintex yielded positive results in four individuals. The results confirm the previously demonstrated higher sensitivity of the Helmintex method compared with the KK method.     

Prolonged fasting in mice: a more sensitive approach to genetic diabetes.

Effects of two different periods of fasting were studied on glucose tolerance and insulin response to glucose in genetically diabetic KK and nondiabetic C57BL/6J mice. Blood sugar levels of the KK mice did not differ markedly from those of the C57BL/6J mice at the fed state or after 8 h fasting. They were, however, significantly higher in the KK mice when fasted for 18 h. The serum IRI levels, which were at least twice as high in the KK mice, decreased more markedly after 18 h fasting. The KK mice showed impaired glucose tolerance after 8 h fasting, which became more pronounced after 18 h fasting. The insulin response to glucose in the KK mice was not altered after an 8-hour fast; it was, however, diminished greatly after an 18-hour fast. These data suggest that prolonged fasting is necessary to detect the diabetic traits in the KK mice. The C57BL/6J mice showed neither impaired glucose tolerance nor diminished insulin response to glucose at both periods of fasting. Studies with the F1 hybrids (KK male X C57BL/6J female), which carry half of the diabetic genes, suggest that the mode of inheritance of diabetes in the KK mice might be polygenic.     

Ubiquitylation of MHC class I by the K3 viral protein signals internalization and TSG101-dependent degradation

The Kaposi's sarcoma-associated herpes virus gene product K3 (KK3) subverts the MHC class I antigen presentation pathway by downregulating MHC class I from the plasma membrane. We now show that KK3 associates with MHC class I molecules and promotes ubiquitylation of class I after export from the endoplasmic reticulum. Ubiquitylation requires the KK3 N-terminal plant homeodomain and provides the signal for class I internalization at the plasma membrane. Once internalized, ubiquitylated MHC class I is targeted to the late endocytic pathway, where it is degraded. Depletion by small interfering RNA of TSG101, a ubiquitin enzyme 2 variant protein involved in late endosomal sorting, prevents class I degradation and preserves cell surface class I expression in KK3-expressing cells. These results suggest a mechanism by which the KK3-induced class I ubiquitylation provides a signal for both internalization and sorting to the late endosomal pathway for degradation. KK3 is the first viral gene product that subverts the trafficking of a host protein via the ubiquitin-dependent endosomal sorting machinery.     

Angiopoietin-like protein 3, a hepatic secretory factor,activates lipolysis in adipocytes

Our previous work identified a genetic mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/Snk mice (previously KK/San), a mutant strain of KK obese mice. KK/Snk had significantly lower plasma triglyceride and free fatty acid (FFA) than KK mice. Human ANGPTL3 treatment increased both plasma triglyceride and FFA. ANGPTL3 inhibited the activity of lipoprotein lipase, which accounted for the increase of plasma triglyceride. The mechanism how ANGPTL3 affects plasma FFA has not been known. The current study reveals that ANGPTL3 targets on adipose cells and induces lipolysis. Both plasma FFA and glycerol decreased in KK/Snk and increased by the treatment of human ANGPTL3. Specific bindings of ANGPTL3 to adipose cells were shown using fluorescence-labeled protein visually and 125I-labeled protein by the binding analysis. Furthermore, ANGPTL3 activated the lipolysis to stimulate the release of FFA and glycerol from adipocytes. We conclude that ANGPTL3 is a liver-derived lipolytic factor targeting on adipocyte.     

Effect of chronic metformin treatment of hepatic and muscle glycogen metabolism in KK mice.

Noninsulin-dependent diabetic KK mice, aged 90-100 days, with hyperinsulinemia and insulin resistance were treated with either metformin (N = 13) or water (control, N = 10) orally at a concentration of 50 mg/kg twice daily for 28 weeks. Age-matched nondiabetic Swiss Webster (SW) mice were also similarly treated. Liver and skeletal muscle glycogen synthase and phosphorylase enzymes were determined in all groups of mice. Both enzymes were significantly lower in control KK than in control SW mice. Metformin did not influence either of these enzymes in nondiabetic SW mice. However, it significantly increased the active form of glycogen synthase (a form) in both the liver and muscle of KK mice. Metformin also increased the active form of phosphorylase (a form) in the liver but not in the muscle of these mice. Hepatic glycogen content was similar in both control and metformin-treated KK mice. However, the muscle glycogen content was significantly higher in metformin-treated than in control KK mice. These data suggest that metformin preferentially stimulates glycogen synthesis in skeletal muscle, and this seems to be responsible for the observed improvement in fasting glucose and glucose response to an oral glucose load in KK mice.     

ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.     

两种自发性2型糖尿病小鼠生物学特性比较

目的分别以C57BL/6JSlac和C57BL/KsJ-db/＋表型正常小鼠为对照组,比较自发性2型糖尿病KK-Ay/Ta和C57BL/KsJ-db/db小鼠的体生长曲线、糖代谢曲线、血清胰岛素水平、主要脏器重量、脏器系数等生物学特性的差异,并探讨其肾脏、肝脏和胰腺等组织病理学变化。方法在各自实验周期内,每2周测定实验组和对照组小鼠的体重、血糖以及血清胰岛素水平,实验结束后处死,脏器、脂肪称重,部分组织制作病理切片。结果 （1）KK小鼠体重远高于db/db小鼠,且同品系间雄性小鼠重于雌性小鼠（P〈0.05）;（2）同品系间雄性小鼠的血糖值明显大于雌性小鼠（P〈0.05）,db/db小鼠出现血糖异常症状比KK小鼠早,且血糖值大于KK小鼠（P〈0.05）,而KK小鼠血糖异常持续时间则较db/db小鼠长;（3）KK小鼠的血清胰岛素水平明显高于db/db小鼠（P〈0.05）,同品系雌雄小鼠间没有明显差异（P〉0.05）;（4）雄性KK小鼠脂肪系数及部分脏器萎缩程度大于雌性,而db/db小鼠雌雄间则无明显差异（P〉0.05）,同时db/db小鼠脾脏和胰腺的萎缩程度及脂肪系数大于KK小鼠（P〈0.05）,而KK小鼠肝脏的萎缩程度则大于db/db小鼠;（5）糖尿病模型小鼠肾脏、肝脏以及胰腺组织均出现明显病变。结论 KK-Ay/Ta和C57BL/KsJ-db/db小鼠均是肥胖的,伴有高血糖、高度胰岛素抵抗,肝脏、肾脏病变和胰岛功能不足的适用性2型糖尿病动物模型,且db/db小鼠血糖出现异常比KK小鼠早、脂肪系数大,而KK小鼠血糖异常持续时间较db/db小鼠长,同时血清胰岛素水平远大于db/db小鼠。