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Police football sessions credited with helping reduce anti social behaviour in Ewloe and Hawarden
Published: Monday, May 14th, 2018
NOTE: This content is old - Published: Monday, May 14th, 2018.
Anti-social behaviour by teenagers which has blighted two areas of Deeside is on the decline thanks to a police initiative supported by a Wales & West Housing Association.
Officers from the South Flintshire Policing team came up with the idea of Friday night football sessions following growing reports of anti-social behaviour by youngsters in the area which included criminal damage and egg throwing at cars.
Wales & West were also victims of criminal damage after the office complex car park barriers were vandalised by a group of youths.
More than 20 youngsters aged 12-17 now attend a weekly football session at Hawarden High School run by police community support officer Stephanie Jones and Dan Williams, school and community sports coordinator from Aura Leisure and Libraries.
PCSO Stephanie Jones said: "Myself and Dan both felt that Hawarden and Ewloe were lacking positive activities that could be utilised by local young people to entertain their time more constructively outside of school.
"Wales & West Housing generously provided £500 of the £750 raised to support the programme, with the rest provided by Flintshire County Council's ASB tasking committee and Aura Leisure and Libraries.
"The football sessions have definitely had a positive impact on the levels of Anti-Social Behaviour in the area, particularly on Friday evenings.
"Prior to the programme, ASB in Hawarden and Ewloe was a monthly point of discussion at police meetings based on the number of reports we were receiving but six months into our project the area has been removed from the agenda, demonstrating the vast improvement that has occurred."
Many of the anti-social behaviour suspects blamed their actions on boredom and having nothing to do after school. But with the regular activity in place since September last year ASB crime in Ewloe and Hawarden is declining – and relations between the police and young people are on the way up.
Wales office of Wales & West Housing Chief executive Anne Hinchey said: "Our sponsorship programme is focused on identifying projects and organisations which match our own vision of making a difference within the community so we are delighted to see the positive impact which the football sessions have had in the area."
PCSO Jones now hopes to extend the project and expand it in future. She added: "On some of the busiest weeks as many as 50 have attended to take part, which is fantastic.
"Dan and I are present at every session as I feel it's important to remove barriers between young people and the police, eliminating their preconception of what we do."
The football sessions take place every Friday evening from 6pm-7pm at Hawarden High School outdoor pitch and are free to attend for anyone aged 12-17.
No advanced booking is needed. Sessions are operated on a drop-in basis so players can just turn up on the night.
Morning - Dry with sunny spells early on but some cloudy knocking about - There'll be an increased risk of showers later this afternoon according to forecasts - Heavier rain possible this evening - High 21º ... PIC:

redpajama_set_name: RedPajamaCommonCrawl


Part 2 of the Housing (Wales) Act 2014 (HWA 2014) makes provisions in relation to homelessness. Chapter 1 of Part 2 explains that there is a duty on local housing authorities to carry out a homelessness review and formulate a homelessness strategy based on the results of that review. The review must include a review of - (a) the levels, and likely future levels, of homelessness in the local housing authority area, (b) the activities carried out in the local housing authority's area that aim to: (i)prevent homelessness,(ii) ensure that suitable accommodation is or will be available for those who are homeless, and (iii) ensure that satisfactory support is available to those who are homeless. In addition, a review of the resources available to the authority must be included within a review. The results of this review must be published, and made available on the local housing authority's website, to any member of the public who asks for a copy, and at the principal office for inspection at all reasonable hours (section 51(2)).
In 2018, a new homelessness strategy must be adopted by a local housing authority, and a new strategy in every fourth year after 2018 must then be adopted (section 50 (2)).
Under section 52, a homelessness strategy, is a strategy with the following objectives: preventing homelessness, providing suitable accommodation and providing satisfactory support to people who are or may become homeless, within the local authority's housing area. More detailed objectives may be pursued, and a strategy may also include provision relating to the specific action which the authority expects to be taken by any public authority or voluntary organisation, who are capable of contributing to the achievement of those objectives, subject to the approval of the body or person concerned (section 52(3)). A homelessness strategy must also include details on action to be taken in relation to individuals who may be in particular need of support. These include people who are leaving prison, care, the armed forces and hospital after receiving medical treatment for mental disorder as an inpatient. People receiving mental health services in the community may also be in particular need of support.
A local authority must consult other relevant bodies before a strategy is adopted or modified, and the strategy should be published in the same way as a review is published, by being made available to members of the public (section 52(9)). The strategy must then be kept under review (section 52(7)).
Chapter 2 places certain duties on local authorities to help people who are homeless or threatened with homelessness in certain circumstances. In assessing its duty the local authority will have to take into account eligibility, homelessness, priority need, intentional homelessness and local connection. The introduction of WA 2014 has the effect that the Housing Act 1996 (Part 7) which previously applied to Wales, now applies only to England. There is considerable case law in relation to homelessness, this paragraph focuses on the relevant legislation.
Section 61 of HWA 2014 states that Schedule 2 to the Act determines whether an applicant is eligible for help. Paragraph 1(1) of Schedule 2 provides that a person is not eligible for assistance under Part 2 of the Act, if he or she is a person from abroad who is ineligible for housing assistance. Subsection(2) provides that a person who is subject to immigration control within the meaning of the Asylum and Immigration Act 1996 is not eligible for housing assistance unless he or she is of a class prescribed by regulations made by the Welsh Ministers. The function of making regulations was originally a function of the Secretary of State but has transferred to the Welsh Ministers. However, this function is exercisable concurrently with the Secretary of State in relation to Wales. This means either the Welsh Ministers or the Secretary of State may exercise the function. In general 'other persons from abroad', i.e. those who are not subject to immigration control, are eligible for housing assistance, unless they fall within a class prescribed by the Welsh Ministers as ineligible (see paragraph 1(4)). The Allocation of Housing and Homelessness (Eligibility) (Wales) Regulations 2014 are the applicable regulations in Wales.
Section 60 of HWA 2014 provides that a local authority must secure that information and advice about homelessness and the prevention of homelessness is available free of charge to any person in its area. The service offered by a local authority must include, in particular, the publication of information and advice on the system provided for by Chapter 2 and how the system operates; whether any help for people who are homeless or may become homeless is available in the authority's area; and how to access the help that is available (section 60(2)). The local housing authority must work alongside other public authorities, voluntary organizations and other persons, to ensure that the service is designed to meet the needs of groups at particular risk of homelessness. Such groups include people leaving prison or youth detention accommodation, young people leaving care, people leaving the regular armed forces, people leaving hospital after medical treatment for mental disorder as an inpatient, and people receiving mental health services in the community (subsection (4)). The service required by section 60 may be integrated with the service required by section 17 of the Social Services and Well-being (Wales) Act 2014, under which local authorities are required to provide information and advice relating to care and support (section 60(6)).
Section 55 of HWA 2014 outlines when a person is homeless or threatened with homelessness. A person is homeless if they, together with anyone who normally resides with them, have no accommodation in the UK or elsewhere which they have a legal right to occupy. A person is also homeless if he or she has accommodation but cannot secure entry to it, or the accommodation is a moveable structure, vehicle or vessel adapted for human habitation (such as a caravan or house boat) and there is no place where it can be placed in order to provide accommodation.
A person shall not be treated as having accommodation unless it is accommodation which it would be reasonable for that person to continue to occupy. The Code of Guidance for Local Authorities on Allocation of Accommodation and Homelessness (Code of Guidance) provides guidance to local authorities on when it might not be reasonable to occupy accommodation, and there is also case law on this subject.
Section 57 of HWA 2014 also specifically provides that it is not reasonable for a person to occupy accommodation if it is probable that this will lead to the person, or a member of the person's household, being subjected to abuse. A member of the person's household includes someone who normally resides with them as a member of his or her family, or any other person who might reasonably be expected to reside with them.
For the purposes of section 57, "abuse" means physical violence, threatening or intimidating behaviour and any other form of abuse which, directly or indirectly, may give rise to the risk of harm. "Domestic abuse" occurs where the victim is associated with the abuser (section 58). The Housing Act 1996 previously referred to "violence"; but this has now been changed to "abuse" to ensure that the Act is not restricted to physical violence.
Section 57(3) of HWA 2014 also provides that in determining whether it is reasonable for a person to continue to occupy accommodation, local authorities may have regard to the general housing circumstances prevailing in the area. A Local Authority may also have regard to whether or not accommodation is affordable for that person. The Code of Guidance states that this could include considering physical conditions, overcrowding, and the type of accommodation.
A person is threatened with homelessness if it is likely that the person will become homeless within 56 days (section 55(4) of HWA 2014).
Section 64 outlines the ways which a local housing authority may secure or help to secure that suitable accommodation is available for occupation by an applicant. A local authority may arrange for a person other than the authority to provide something, or it may itself provide something. Alternatively, the local authority may provide something, or arrange for something to be provided to a person other than the applicant (section 64(1)). Section 64(2) provides a list of examples of what may be provided or arranged. The examples include mediation, payments by way of grant or loan, guarantees that payments will be made, advocacy and accommodation. In addition to this, the Code of Guidance includes a list of interventions that Local Authorities ought to have in place as a minimum set of available interventions (chapter 12.13). This non-exhaustive list includes domestic abuse services, employment and training option advice, specialist welfare services for armed forces personnel, action to intervene with mortgage areas and action to support disabled applicants. Local Authorities should not be restricted to this list, and other innovative interventions based on local demand must also be identified and considered in Local Homelessness Strategies. Local Authorities must be pro-active in their utilisation of this range of interventions, and must offer support for applicants on an individual basis. Along with the local authority's duty to be pro-active, the Welsh Ministers are also under a duty to give guidance to local housing authorities in relation to how they may secure or help to secure suitable accommodation for an applicant.
The meaning of 'help to secure' is defined under section 65. The authority must take reasonable steps to help, having regard to the need to make the best use of the authority's resources. But the authority is not required to secure an offer of accommodation under Part 6 of the Housing Act 1996, or to otherwise provide accommodation. Paragraph 12.7 of the Code of Guidance emphasizes that where a duty under section 66 applies, a Local Authority has a duty to help an applicant, but this does not mean that the Local Authority has a duty to secure accommodation.
The duty under section 66 of HWA 2014 provides that a local authority must help to secure that suitable accommodation does not cease to be available for occupation if the authority is satisfied that the applicant is threatened with homelessness, and is eligible for help. The duty to help to secure accommodation under section 66 ends when the circumstances specified in section 67 arise.
There is also a duty under section 73 of HWA 2014 to help to secure accommodation for homeless applicants who are eligible for help. The circumstances in which the duty under section 73 ends are set out in section 74.
Where a person presents as homeless or threatened with homelessness to the local authority, the local authority may have a duty to provide interim accommodation whilst it considers a person's application and decides what duty, if any, it owes to the person. Section 68 of HWA 2014 provides that interim accommodation must be provided if the local authority has reason to believe that an applicant may be homeless, eligible for assistance and have a priority need for accommodation (subsection (2)).
Interim accommodation is often offered in the form of B&B accommodation, hostels or refuges whilst the local authority conducts its investigations.
Whether or not a person has priority need affects the duty the local authority will have in relation to that person. As explained above, in order for the duty to provide interim accommodation to apply to an applicant, the local authority must have reason to believe the applicant has priority need, amongst other things. The main homelessness duties in section 75 of HWA 2014 also apply only to applicants who have a priority need for accommodation.
i) a person who has a local connection with the area and is vulnerable as a result of being an ex-prisoner.
A person who resides, or might reasonably be expected to reside with any of the above.
Note that a person who is not eligible for assistance under Part 2 of HWA 2014 is not to be taken into account when assessing priority need, for example, a dependent child who was subject to immigration control and ineligible would not be classed as a dependent child for the purpose of assessing priority need. The Welsh Ministers can add or remove descriptions of persons having priority need by order under section 72.Chapter 16 of the Code of Guidance relates to priority need.
Section 71 of HWA defines who is vulnerable under section 70(1)(c) and 70(1)(j). Section 70(1)(c) relates to those of old age, mental illness or handicap or physical disability. Whereas section 70(1)(j) relates to ex-prisoners. Section 71 states that a person who is vulnerable would be less able than an ordinary homeless person to fend for himself or herself, if the person were to become street homeless. Additionally, a person who is vulnerable would suffer more harm than an ordinary homeless person would suffer. There is no equivalent definition of vulnerability contained in English legislation, and the principles established by case law are relied upon instead.
Whether or not a person is intentionally homeless will affect which duties are owed to that person under HWA 2014. There are two ways under the Act in which a person can become homeless, or threatened with homelessness intentionally: (i) by deliberate act or omission (s77(2)). This means that they must have done or failed to do something deliberately to cause their homelessness when they should have known the consequences, such as surrender a suitable tenancy or be evicted for anti-social behaviour. Alternatively: (ii) by entering into an agreement to cease occupying accommodation which it would have been reasonable for them to continue to occupy (s77(4)).
By virtue of section 78(2) of HWA 2014, when assessing an applicant for help with homelessness, a local housing authority may not have regard to whether or not the applicant has become homeless intentionally for the purposes of sections 68 and 75 unless it has decided to have regard to one or more of the categories of applicants specified by the Welsh Ministers. Section 78(1) of the Act places an obligation on the Welsh Ministers to make regulations to specify such categories. The explanatory note to the Homelessness (Intentionality) (Specified Categories) (Wales) Regulations 2015 outlines these duties.
The Welsh Ministers must make regulations for local authorities to have regard to intentionality for certain categories of person. It is intended that local authorities will be allowed to opt to apply the intentionality test for some or all applicants, for certain specified priority need categories. For this exception to take effect, two requirements are necessary: firstly, an applicant must be within a specific category under the Homelessness (Intentionality) Regulations, where it has been agreed that the authority should have regard to whether or not applicants have become homeless intentionally. The second requirement is that the authority has published a notice of its decision specifying the category or categories of applicants in relation to which they will consider intentionality. Welsh Ministers must receive written notice of the local authority's decision at least 14 days before it takes effect.
Section 96(4) is relevant when a local housing authority rules that an applicant who lives with a child, is ruled to be ineligible to receive help as a result of becoming homeless intentionally or becoming threatened with homelessness intentionally. Under such circumstances the council of a county must ensure that its housing department provides the social services department with such assistance as the social services department may reasonably request (see Part 3 of Social Services and Well-Being (Wales) Act 2014).
Whether or not a person has a local connection will affect the local authority's ability to refer the application to a different authority. Sections 80 and 81 of HWA 2014 relate to local connection. In general, where a person has a priority need and is not intentionally homeless the authority must accept the duty to accommodate that person under section 66 of HWA 2014 if they have a local connection. If the person has no local connection with the authority's area, the authority can refer the application to an authority where the person does have a local connection. A person may have a local connection with the area of a local housing authority if the person is living or has lived there, is working in the area, because of family reasons or because of special circumstances (s81(2)).
Once an authority has completed its enquiries it must notify the applicant of its decision and whether any duties are owed (section 63 of HWA 2014). Applicants have the right to request a review of the decision within 21 days of being notified of certain decisions of the authority (section 85 of HWA 2014). The procedure to be followed is to be set out in regulations made by the Welsh Ministers under section 86 of HWA 2014. The current regulations are the Homelessness (Review Procedure) (Wales) Regulations 2015/1266.
The 'main duty' arises in relation to a person who is eligible for assistance, homeless, has a priority need, is not intentionally homeless and is not subject to a local connection referral. The main duty is to provide suitable accommodation (section 75 of HWA 2014) until the authority ceases to be subject to the duty (section 76). Certain actions specified in section 67 trigger the cessation of the duty. This includes, amongst other things, where a person refuses certain offers of accommodation, and where the accommodation offered is likely to be available for a period of at least 6 months.


How can I make percentage rates in Pro Sites?
I am looking for a new payment solution for Pro Site. Normally, customers can pay a fixed monthly amount. You have the opportunity to stagger that monthly.
But I want only a variable monthly amount.
This derives from the payment options.
First and foremost I am thinking of Market Press. I want for example five percent of the sales from my clients.
Thus requires a separate entry field in the database and in the pro site. Market Press lists the sales in the database with the date.
,name and address of me and the user ,a period description and services sales.
Also a requested amount including VAT. That bill solution seems to be possible with Pro SITE very easy by editing.
The amount appeared then at the end of the month as a buttonfield with text. The customer is obliged to pay this. As with Pro Site when you can start a monthly payment. If the client is not paying then market press is for him and the customer no longer visible.
I need the option of variable payments according to percentages in Pro Site. These arise from the monthly sales of the stores.
The answer seems to be in the marketpress-stats and in the payment modul in Pro Site.
Then I ask just different.
print the total sales of the affected blog?
In which document will these values ??are printed out?
How could the link look like from a percentage value instead of the prices in combination with the value of total sales to be printed out as a value?
Where is this value passed to the gateway?
This won't be as simple as changing the price of the level periodically. You will actually need to create a brand new payment gateway for this.
The problem is, a customer's subscription payment will always be based on the price from when they signed up. This means a user can sign up at $10, and continue to receive that rate as long as the subscription remains active. You could even raise their level to a price of $20, but they'd still be paying $10 - unless they cancelled and created a new subscription, or changed plans.
So you would need to create a gateway that sets a balance on their account based on your variables.
You're welcome! This has been added to feature requests, so we can consider it for future development. We're also keen to hear what other community members think.


The use of low-level electrical stimulation for pain relief is not a new treatment. It has been used for years by athletic trainers, chiropractors, and physical therapists to relieve pain from injuries and chronic muscle and skeletal discomfort. Those treatments are administered in an office using large machines.
The Painmaster patches look like the electrodes used in a doctor's office for administering electrocardiograms. They are applied and removed just as effortlessly.


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Preventive treatment with liraglutide protects against development of glucose intolerance in a rat model of Wolfram syndrome
Maarja Toots2 na1,
Kadri Seppa1 na1,
Toomas Jagomäe1,
Tuuliki Koppel2,
Maia Pallase2,
Indrek Heinla2,
Anton Terasmaa ORCID: orcid.org/0000-0002-5139-17641,
Mario Plaas1,2 &
Eero Vasar1,3
Scientific Reports volume 8, Article number: 10183 (2018) Cite this article
Endocrine system and metabolic diseases
Wolfram syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WFS1 (Wolframin1) gene. The syndrome first manifests as diabetes mellitus, followed by optic nerve atrophy, deafness, and neurodegeneration. The underlying mechanism is believed to be a dysregulation of endoplasmic reticulum (ER) stress response, which ultimately leads to cellular death. Treatment with glucagon-like peptide-1 (GLP-1) receptor agonists has been shown to normalize ER stress response in several in vitro and in vivo models. Early chronic intervention with the GLP-1 receptor agonist liraglutide starting before the onset of metabolic symptoms prevented the development of glucose intolerance, improved insulin and glucagon secretion control, reduced ER stress and inflammation in Langerhans islets in Wfs1 mutant rats. Thus, treatment with GLP-1 receptor agonists might be a promising strategy as a preventive treatment for human WS patients.
Wolfram syndrome (WS) is a rare neurodegenerative disorder caused by biallelic mutations of the Wolframin1 (WFS1) gene and whose main symptoms are diabetes mellitus, optic nerve atrophy, hearing loss, and neurodegeneration in the brainstem1,2,3. Unfortunately, no effective cure exists for WS, and currently treatment is focused on relieving symptoms4. Corrupted function of WFS1 leads to impairment of cellular calcium regulation and increase in endoplasmic reticulum stress, ultimately causing cell death. Thus, one possible strategy for treatment of WS is to normalize cellular calcium regulation and reduce endoplasmic reticulum stress4.
GLP-1 (glucagon-like peptide-1) receptor agonists have been accepted as a promising class of anti-diabetic drugs, having the potential to delay or even reverse disease progression5. An incretin peptide, GLP-1 regulates glucose homeostasis, metabolism, and neural survival directly on tissues expressing GLP-1 receptors and indirectly through neuronal and endocrine pathways6,7. Acute treatment with the GLP-1 agonist exenatide has shown a promising anti-diabetic effect in Wfs1 knock-out mice8. The GLP-1 agonist Exendin-4 was shown to reduce endoplasmic reticulum (ER) stress in cardiac myocytes and prevent a decrease in activity of sarco/endoplasmic reticulum Ca-ATPase-2a (SERCA2a)9. Early intervention with the GLP-1 receptor agonist liraglutide improved glucose tolerance and beta cell apoptosis in prediabetic Gato-Kakizaki rats10. Considering these results, treatment with GLP-1 agonists appears to be an efficient way to reduce elevated ER stress in vitro and in vivo. Thus, GLP-1 agonists could be a feasible strategy for managing WS.
Recently, a rat model of WS has been developed and validated11. Deletion of exon 5 of the Wfs1 gene resulted in development of the main symptoms of WS: diabetes mellitus (Fig. 1), medullary degeneration, and optic nerve atrophy11. Moreover, levels of ER stress were elevated in the pancreas and brainstem of this rat11. The progressive development of diabetes mellitus and other WS symptoms makes the Wfs1-ex5-KO232 rat a good tool to study treatment options. Therefore, the aim of the present study was to investigate the effect of chronic treatment with GLP-1 receptor agonist on the progression of glucose intolerance in a Wfs1 mutant rat.
WS development in Wfs1 deficient rat. Based on Plaas et al.11.
One-week treatment with liraglutide restores glucose tolerance in 5-month-old Wfs1 KO rats
In accordance with previous findings11, 5-month-old Wfs1 KO (Wfs1 exon 5 knock-out) rats were glucose intolerant, with peak blood glucose levels over 20 mM in response to glucose administration (Fig. 2a). Before liraglutide treatment, Wfs1 KO animals showed glucose intolerance (Fig. 2b) (F(1,12) = 28.28, p < 0.001 (genotype); F(1,12) = 63.67, p < 0.001 (treatment); F(1,12) = 26.16, p < 0.001 (genotype × treatment)); however, after 8-day repeated treatment with 0.4 mg1kg−1day−1 liraglutide, the difference between the genotypes disappeared (Fig. 2a); area-under-curve analysis confirmed this finding (Fig. 2b) (p > 0.05). This indicates that liraglutide improved glucose tolerance in Wfs1 KO rats (Fig. 2a,b).
Repeated liraglutide effect on glucose intolerant 5-month-old Wfs1 KO rats. (a) IPGTT (2 g/kg) before and after 8-day chronic administration of 0.4 mg/kg liraglutide to 5-month-old male Wfs1 KO and control rats. (b) Decrease in IPGTT (2 g/kg) area under curve before and after chronic liraglutide treatment for 8 days. Glucose-stimulated increases in blood (c) insulin and (d) C-peptide levels before and 30 minutes after glucose administration. The data were compared using repeated measures ANOVA followed by Tukey's HSD tests; £££p < 0.001 compared to WT AUC during same IPGTT. ***p < 0.001 compared to baseline of the same genotype during the same IPGTT. ##p < 0.01 compared to WT animals in the same timepoint during the same IPGTT. ¤p < 0.05 compared to WT saline animals in the same timepoint during the first IPGTT. The data are presented as the mean ± SEM, n = 6–8.
Glucose-stimulated insulin secretion (Fig. 2c) was significantly weaker in KO animals 30 minutes after glucose challenge (F(1,23) = 6.07, p < 0.05 (genotype); F(1,23) = 17.38, p < 0.001 (timepoint); F(1,23) = 6.84, p < 0.05 (timepoint × genotype)). Both in wild-type (WT) and Wfs1 KO rats, the basal insulin levels were decreased and glucose-stimulated insulin secretion increased after liraglutide treatment (F(1,23) = 0.051, p = 0.82 (treatment); F(1,23) = 0.14, p = 0.71 (genotype × treatment); F(1,23) = 9.01, p < 0.01 (timepoint × treatment); F(1,25) = 0.17, p = 0.686 (timepoint × genotype × treatment)).
Similarly to glucose-stimulated insulin secretion, C-peptide levels (Fig. 2d) were significantly diminished in KO animals 30 minutes after glucose challenge (F(1,21) = 13.10, p < 0.01 (genotype); F(1,21) = 26.95, p < 0.001 (timepoint); F(1,21) = 9.1, p < 0.01 (timepoint × genotype)). Liraglutide decreased basal C-peptide levels and showed tendency toward increased C-peptide levels 30 minutes after glucose challenge, when compared to baseline (F(1,21) = 3.71, p = 0.068 (treatment); F(1,21) = 2.16, p = 0.16 (genotype × treatment); F(1,21) = 9.69, p < 0.01 (timepoint × treatment); F(1,21) = 0.008, p = 0.93 (timepoint × genotype × treatment)).
Administration of liraglutide, from an early age inhibits the development of glucose intolerance in Wfs1 KO rats
As one-week treatment with liraglutide improved the glucose control in already glucose intolerant 5-month-old-rats, we aimed to investigate, if chronic treatment with liraglutide starting before the onset of glucose intolerance would be able to prevent or delay the development of diabetes in Wfs1 KO rats. At 2 months of age, Wfs1 KO rats' glucose tolerance was indistinguishable from WT littermates' (Fig. 3b). For 19 weeks of liraglutide administration, the glucose tolerance profile of Wfs1 KO rats receiving medication remained similar to that of their WT littermates, while Wfs1 KO rats in control group developed glucose intolerance (Fig. 3c–e), demonstrated by stable increase of area-under-curve (Fig. 3f) (F(1,50) = 22.598, p < 0.001 (genotype); F(1,50) = 18.27, p < 0.001 (treatment); F(1,50) = 4.047, p < 0.05 (genotype × treatment); F(3,150) = 24.26, p < 0.001 (age); F(3,150) = 11.77, p < 0.001 (age × genotype); F(3,150) = 19.82, p < 0.001 (age × treatment); F(3,150) = 5.12, p < 0.01 (age × genotype × treatment)).
Development of glucose intolerance over 19 weeks of liraglutide treatment. (a) Weight change over 19 weeks of liraglutide administration. Blood glucose profile after glucose challenge during liraglutide treatment (b) before, (c) 7 weeks, (d) 14 weeks and (e) 19 weeks after the beginning of liraglutide treatment. (f) Area under curve analyses for IPGTT results at different timepoints. (g) Langerhans islet mass and (h) Langerhans islet mass/body weight ratio after 19 weeks of liraglutide treatment. (i) Insulin tolerance test with 1U/kg human insulin after 18 weeks of liraglutide treatment. The data were compared using repeated measures ANOVA or one-way ANOVA followed by Tukey's HSD tests; *p < 0.05, **p < 0.01, ***p < 0.001 compared to (same-age) WT saline-treated animals. ###p < 0.001 compared to same-age Wfs1 KO liraglutide-treated animals. The data are presented as the mean ± SEM, n = 12–15 (weight and IPGTT), n = 6–8 (ITT), n = 4–6 (islet measurements).
Insulin sensitivity (Fig. 3i) after 18 weeks of liraglutide/saline treatment was not different between genotypes (F(1,24) = 3.36, p = 0.079) and was not affected by liraglutide treatment in either genotype (F(1,24) = 4.03, p = 0.056 (treatment); F(1,24) = 0.617, p = 0.44 (genotype × treatment)).
Langerhans islet mass decrease is reduced in liraglutide-treated Wfs1 KO rats
Our next purpose was to assess the effect of chronic liraglutide treatment by measuring the Langerhans islet mass. Langerhans islets were visualized by staining with anti-insulin antibody. The staining effectively labeled islets in every treatment group and genotype (Supplementary Fig. 1). In agreement with a previous study11, Langerhans islet mass in saline-treated Wfs1 KO animals was lower than in saline-treated WT rats (Fig. 3g) (p < 0.01). Similarly to previous reports12, 19 week liraglutide treatment resulted in a significantly decreased islet mass in WT pancreases, compared with WT saline treated animals, but this effect was not present between Wfs1 KO treatment groups (F(1,16) = 7.58, p < 0.05 (genotype); F(1,16) = 1.72, p = 0.208 (treatment); F(1,16) = 9.93, p < 0.01 (genotype × treatment)). This opposite effect of liraglutide on Langerhans islet mass in two genotypes might be attributable to its combined action on body weight and beta cell health. Liraglutide treatment induced a decrease in body weight in both genotypes (Fig. 3a) (F(1,51) = 18.24, p < 0.001 (genotype); F(1,51) = 21.90, p < 0.001 (treatment); F(1,51) = 0.81, p = 0.37 (genotype × treatment)). Liraglutide-induced decrease in the Langerhans islet mass was paralleled by a decrease in body weight; thus, Langerhans islet mass/body weight ratio was not statistically different between liraglutide and saline treated WT rats. However, saline-treated Wfs1 KO rats had significantly lower Langerhans islet mass/body mass ratio (p < 0.05) than saline-treated WT rats (Fig. 3h). This difference was eliminated under liraglutide treatment, bringing Wfs1 KO rats' Langerhans islet mass/body mass ratio similar to that of WT rats (F(1,16) = 2.34, p = 0.15 (genotype); F(1,16) = 0.02, p = 0.89 (treatment); F(1,16) = 8.50, p < 0.05 (genotype × treatment)).
Liraglutide inhibits glucagon secretion induced by glucose stimulation in Wfs1 KO rats
As the decrease of Langerhans islet mass appeared to be reduced in liraglutide treated Wfs1 KO rats, we next measured glucagon and insulin secretion, to assess the responsiveness of islet cells to glucose challenge. For 19 weeks, in liraglutide-treated or control rats, there were no apparent changes in insulin secretion, measured 30 minutes after glucose injection (Fig. 4a–d).
Serum insulin, C-peptide, and glucagon levels before and 30 minutes after glucose administration. (a–d) Insulin levels after various time on liraglutide treatment, (e) C-peptide and (f) glucagon levels after 19 weeks on liraglutide treatment. The data were compared using repeated measures ANOVA followed by Tukey's HSD tests; *p < 0.05, **p < 0.01, ***p < 0.001 compared to the baseline of the same animals; #p < 0.05, ##p < 0.01, ###p < 0.001 compared to WT animals of same treatment at the same timepoint; ¤p < 0.05, ¤¤¤p < 0.001 compared to saline treatment of the same GT at the same timepoint. The data are presented as the mean ± SEM, n = 12–15.
While insulin is rapidly removed from circulation by the liver, the half-life of C-peptide is at least 20 minutes13,14. As insulin and C-peptide are secreted in equimolar concentrations, we can presume that C-peptide concentration measured 30 minutes after glucose challenge reflects the combined secretion of first- and second-phase insulin during the whole stimulatory period. C-peptide basal levels were decreased in liraglutide-treated animals from both genotypes compared to their respective controls (F(1,51) = 12.30, p < 0.001 (treatment)). C-peptide level doubled in WT rats from both treatment groups in response to glucose. In control Wfs1 KO animals, the level of C-peptide was not significantly increased in response to glucose, but in liraglutide-treated Wfs1 KO rats, the serum C-peptide levels doubled 30 minutes after glucose administration when compared to the basal level (Fig. 4e) (F(1,51) = 37.58, p < 0.001 (genotype); F(1,51) = 2.08, p = 0.155 (treatment × genotype); F(1,51) = 202.72, p < 0.001 (timepoint); F(1,51) = 12.28, p < 0.001 (timepoint × treatment); F(1,51) = 27.38, p < 0.001 (timepoint × genotype); F(1,51) = 0.07, p = 0.79 (timepoint × treatment × genotype)).
Glucagon levels were significantly lower 30 minutes after glucose stimulation in liraglutide-treated Wfs1 KO rats than in saline-treated Wfs1 KO rats (Fig. 4f) (F(1,47) = 10.36, p < 0.01 (treatment); F(1,47) = 3.29, p = 0.076 (genotype); F(1,47) = 1.54, p = 0.22 (treatment × genotype); F(1,47) = 0.78, p = 0.383 (timepoint); F(1,47) = 0.81, p = 0.371 (timepoint × treatment); F(1,47) = 4.08, p < 0.05 (timepoint × genotype); F(1,47) = 0.32, p = 0.57 (timepoint × treatment × genotype)).
Liraglutide treatment decreases ER stress, inflammation, and proliferation in Langerhans islets
To further assess the overall health of islet cells, we performed gene expression analyses of Langerhans islets, extracted from rats after 19 weeks on liraglutide/saline treatment. Gene expression revealed no significant changes in Wfs1 expression (Fig. 5a) (F(1,25) = 5.70, p < 0.05 (genotype); F(1,25) = 0.95, p = 0.339 (treatment); F(1,25) = 0.099, p = 0.756 (genotype × treatment)).
Langerhans islet gene expression analyses after 19 weeks of liraglutide treatment compared to WT saline group. Gene expression of (a) Wolframin1; (b) GLP-1 receptor; ER stress markers (c) Grp78 and (d) Xbp1 splicing; inflammation marker (e) IP10; proliferation marker (f) Ki67. The data were compared using factorial ANOVA followed by Tukey's HSD tests; *p < 0.05, **p < 0.01, ***p < 0.001 compared to WT animals of the same treatment; #p < 0.05, ##p < 0.01 compared to saline animals of the same GT. The data are presented as the mean ± SEM, n = 4–8.
GLP-1 receptor (Fig. 5b) expression was decreased in Wfs1 KO animals (F(1,25) = 44.70, p < 0.001). Liraglutide had a slight increasing effect on GLP-1 receptor mRNA levels in both genotypes (F(1,25) = 5.64, p < 0.05 (treatment); F(1,25) = 0.056, p = 0.814 (genotype × treatment)).
The ER stress marker Grp78 (Fig. 5c) and Xbp1 splicing (Fig. 5d) were increased in Wfs1 KO rats compared to WT littermates (F(1,25) = 13.06, p < 0.01 and F(1,16) = 6.13, p < 0.05 respectively). Liraglutide treatment decreased ER stress (F(1,25) = 3.58, p = 0.071 and F(1,16) = 4.94, p < 0.05 respectively), resulting in similar levels in WT and liraglutide-treated KO animals (F(1,25) = 0.095, p = 0.760 (genotype × treatment) (F(1,16) = 0.95, p = 0.343 (genotype × treatment)). Reduction in ER stress was not accompanied by changes in apoptosis markers (Supplementary Fig. 2).
Inflammatory marker IP10 (Fig. 5e) levels in Langerhans islets showed no differences between genotypes (F(1,20) = 0.03, p = 0.860). Liraglutide treatment had a significant decreasing effect on IP10 expression, regardless of genotype (F(1,20) = 11.61, p < 0.01; F(1,20) = 1.405, p = 0.250 (genotype × treatment)).
Proliferation marker Ki67 (Fig. 5f) expression in Wfs1 KO rats was higher than in WT littermates (F(1,18) = 17.49, p < 0.001). Liraglutide treatment decreased Ki67 expression in both genotypes, bringing the expression level in KO animals similar to WT islets Ki67 expression (F(1,18) = 20.22, p < 0.001 (treatment); F(1,18) = 0.35, p = 0.564 (genotype × treatment)).
Currently there is no preventive cure for WS and treatment is limited to management of symptoms4. In our previous studies we have shown that acute treatment with valproic acid normalized glucose intolerance in Wfs1 KO mice15. However, 3 months long chronic treatment with valproic acid did not improve the diabetic phenotype of Wfs1 KO mice16. These results highlight the importance of preclinical evaluation of long term effects of drug treatment, even if the results of short term studies seem promising. Therefore, in this study we evaluated the effects of short-term and long-term treatments with GLP-1 receptor agonist on blood glucose control in Wfs1 KO rats. The aim was to investigate whether chronic treatment with the GLP-1 receptor agonist liraglutide, starting before the onset of metabolic symptoms, would be able to prevent or delay the development of diabetes in Wfs1 KO rats. Our results indicate that chronic liraglutide treatment prevented the development of glucose intolerance in Wfs1 KO rats (Fig. 3f). Moreover, liraglutide reduced ER stress and inflammation in Wfs1 KO rats Langerhans islets (Fig. 5), which seems to result from overall better health of islet the cells and a possible protective effect of liraglutide.
Liraglutide treatment decreased basal insulin and C-peptide levels. When compared to basal levels, liraglutide-treated Wfs1 KO rats showed a twofold increase in both insulin and C-peptide levels 30 minutes after stimulation, while saline-treated Wfs1 KO rats' C-peptide levels did not change from the basal level in response to glucose (Figs 2c,d and 4d,e). While insulin is rapidly removed from circulation by the liver, the half-life of C-peptide is at least 20 minutes13,14. Therefore, we can presume that C-peptide concentration measured 30 minutes after glucose challenge reflects the combined secretion of first- and second-phase insulin during the whole stimulatory period. These results indicate constant low-level insulin secretion from Wfs1 KO rats that is only slightly elevated by glucose challenge. Liraglutide treatment appears to correct that deficit by enhancing the first phase of insulin secretion. In addition to enhancement in insulin secretion, GLP-1R agonists have also been shown to reduce proinsulin:insulin ratio (elevated in subjects with type 2 diabetes), possibly as a result of reduced ER stress and therefore, improved proinsulin processing17. Increased proinsulin:insulin ratio has been shown in mice lacking functional Wfs118. Improper proinsulin processing may also explain the lack of blood glucose lowering effect of depolarizing medication in Wfs1 deficient mice8. As both insulin and C-peptide ELISAs used in current study may also cross-react with proinsulin (according to our inquiry to the manufacturer), the improved glycemic control may also in part result from properly processed insulin after liraglutide treatment. These results are in accordance with those obtained from Wfs1 deficient mice, whose blood glucose levels during IPGTT are significantly lowered after treatment with the GLP-1 analogue exenatide, an effect not accompanied by increased serum insulin concentration 30 minutes after stimulation8. Also, administration of exenatide to Wolfram syndrome 2 (WS2, CISD2 (CDGSH Iron Sulfur Domain 2) mutation) patient lowered daily insulin dose 70% and improved glycemic control19. Knock-down of CISD2 by shRNA in INS-1 cells decreased insulin secretion in response to glucose or KCl by 30% and this effect was also reversed by exenatide treatment19. The similar response in these WS and WS2 models indicates the effectiveness of GLP-1 analogues, probably resulting from the same molecular mechanism.
The change in first-phase insulin secretion may also be at least partially responsible for the change in glucagon secretion (Fig. 4f), as intra-islet insulin, secreted from beta cells, is necessary for glucagon secretion inhibition20,21,22. Also, both liraglutide and GLP-1 have been shown to decrease glucagon levels23,24. Exenatide experiments with Wfs1 KO mice did not reveal any differences in glucagon secretion, but this might be due to a shorter treatment protocol or the fact that Wfs1 KO mice do not exhibit such strong progression of glucose intolerance over time8. Disturbances in suppression of mealtime glucagon concentration are also present in type 2 diabetes patients, whose hepatic glucose production contributes to hyperglycemia25,26. Changes in both insulin (as indicated by C-peptide levels) and glucagon secretion in Wfs1 KO rats are corrected by liraglutide treatment, indicating improved beta and alpha cell sensitivity and function.
Langerhans islet mass doubles in WT rats between the ages of 3 to 7 months while starting to decline in Wfs1 KO rats11. This decline is most likely caused by loss of beta cell mass, as in mouse model of WS, there is 50% decline in beta cell mass between 8 and 36 weeks of age, but this decline is not represented in other cell types of islet27. There are controversial reports regarding GLP-1 and its agonists' effects on beta cell mass12,28,29. In liraglutide-treated WT rats, we saw an approximately 40% decline in Langerhans islet mass (Fig. 3g), which was very similar to the liraglutide response seen in a mouse model on hyperglycemia12. Interestingly, in Wfs1 KO rats, liraglutide had no decreasing effect on Langerhans islet mass, but actually seemed to prevent the decline in Wfs1 KO Langerhans islet mass, as established in our previous study11. Furthermore, when considering the differences in body weight, liraglutide treatment brought Wfs1 KO rats' Langerhans islet mass closer to that of WT animals (Fig. 3h).
Even though decline in Langerhans islet mass did not occur in liraglutide-treated Wfs1 KO rats, Langerhans islet cell proliferation had significantly declined in both genotypes (Fig. 5f), indicating that the main effect in Langerhans islet mass was not caused by increased proliferation in Wfs1 KO rats. These results are in accordance with those obtained from prediabetic Goto-Kakizaki rats, whose proliferation marker Ki67 levels had also declined in response to liraglutide treatment10. In humans, beta cell proliferation is very low after the first few years of life, and treatments increasing proliferation in rodents are unlikely to cause the same in human beta cells30. Therefore, as liraglutide's positive effect appears not to be caused by increased proliferation, it remains a promising treatment strategy for human WS patients.
Unlike Luo et al., we did not see a significant effect of liraglutide on islet apoptosis (Supplementary Fig. 2)10. This might be due to the fact that Luo et al. used very young rats, whose beta cells are actively proliferating and a shorter treatment protocol10,31. Also, reduction in beta cell mass in Wfs1 KO rat develops very slowly over time, as fasting hyperglycemia is apparent around 13 months of age, and even then, most likely there is some portion of beta cells left11. Moreover, we are analyzing whole islets, making it more difficult to detect differences only apparent in beta cells. In healthy rats, 20–35% islet cells are not beta cells and this ratio is even higher in aging Wfs1 KO rats32, who lose beta cell mass. Therefore, it is possible that the difference in rate of apoptosis is so low, that we are not able to detect it. Although there was no significant effect on apoptosis, both ER stress (Fig. 5c,d) and inflammation (Fig. 5e) were reduced by liraglutide treatment. Inflammation has long been recognized as a contributing factor in the development of diabetes33,34, and both GLP-1 agonists and DPP-4 inhibitors have been shown to suppress inflammation in pancreatic islets and adipose tissue35,36,37. Taken together, these results indicate better overall health of islet cells.
Diabetes mellitus is one of the first symptoms of Wolfram syndrome and is also present in a WS rat model. In this study, we demonstrated that one-week treatment with liraglutide improved glucose control in 5-month-old Wfs1 KO rats, who had already developed glucose intolerance by that age. Moreover, we have demonstrated that 19-week long treatment with Liraglutide, starting at young age before manifestation of metabolic symptoms, prevents or delays the development of diabetic phenotype in Wfs1 KO rats. As WS comprises various symptoms, starting with diabetes and eventually leading to neurodegeneration, a lifelong experiment with GLP-1 receptor agonist should follow to assess its possible positive preventive effects on other complications accompanying WS.
Generation and phenotype of a Wfs1 mutant (Wfs1 exon 5 knock-out) rat has been described previously11. Breeding and genotyping were performed at the Laboratory Animal Centre, University of Tartu. For this study, 2- to 7-month-old and 5-month-old male homozygous Wfs1-deficient and wild-type (WT) littermate control rats were used. The animals were housed in cages in groups of 2–4 animals per cage under a 12 h light/dark cycle (lights on at 7 a.m.). Rats had unlimited access to food and water except during testing. Sniff universal mouse and rat maintenance diet (Sniff #V1534) and reverse osmosis-purified water were used. Experiments were performed between 9 a.m. and 5 p.m. Tissue collection was performed as described previously11. Permission for this study was given by the Estonian National Board of Animal Experiments (No 103, 22nd of May 2017) in accordance with the European Communities Directive of September 2010 (2010/63/EU).
Intraperitoneal glucose tolerance tests (IPGTT)
Animals were deprived of food for 3 h before and during the experiment; water was available throughout the experiment. D-Glucose (Sigma-Aldrich) was dissolved in 0.9% saline solution (20% w/vol) and administered intraperitoneally at a dose of 2 g/kg of body weight. Blood glucose levels were measured at the indicated time points from the tail vein using a handheld glucometer (Accu-Check Go, Roche, Germany). Blood samples were drawn from the tail vein immediately before and 30 min after glucose administration for further analyses.
Preliminary study: 8-day repeated liraglutide treatment
In this experiment, 5-month-old rats were used. After the first IPGTT test, the rats received 0.4 mg/kg liraglutide (Novo Nordisk, Denmark) subcutaneously for 8 days. Injections of 1 ml/kg volume were administered once a day between 8 and 11 a.m. The second IPGTT was performed 4.5 hours after the last liraglutide injection.
Chronic liraglutide treatment
The rats were 2 months old at the beginning of the experiment. After the first IPGTT test, the rats were randomly allocated into the liraglutide or control group. The liraglutide group animals received 0.4 mg/kg liraglutide (Novo Nordisk, Denmark) and the control group animals a 0.9% saline solution (vehicle) subcutaneously. Injections of 1 ml/kg volume were made once a day between 8 and 11 a.m. (or immediately after a glucose/insulin tolerance test). Rats were weighed once a week. Glucose tolerance and insulin tolerance tests were performed 24 hours after the previous liraglutide/saline injection. After the last IPGTT, rats were equally allocated for subsequent histological/qPCR analysis according to their IPGTT results.
Insulin, C-peptide, and glucagon measurements
For serum separation, blood was allowed to clot, centrifuged for 15 min at 2000 × g, and stored in −80 °C until further analysis. Serum insulin levels were measured using an ultra-sensitive rat insulin ELISA kit (CrystalChem cat# 90060), C-peptide levels were measured using rat C-peptide ELISA (CrystalChem cat# 90055), and glucagon levels were measured using rat glucagon ELISA (CrystalChem cat# 81519), according to the manufacturer's instructions.
Insulin tolerance tests
An insulin tolerance test was performed one week before the last IPGTT in the same manner as the glucose tolerance tests except that human insulin (1 unit/kg s.c., Lantus Solostar) was administered s.c. instead of glucose.
Determination of Langerhans islet mass
Langerhans islet mass was estimated as has been described previously38. In brief, rats were anaesthetized with an i.p. injection of ketamine (100 mg/kg) and dexmedetomidine (250 µg/kg) and were then perfused transcardially with 4% paraformaldehyde (PFA, Sigma-Aldrich) in 0.1 M phosphate buffer (PB, pH 7.4). Their pancreases were dissected, and excess fat removed. The weight of each pancreas was recorded, and the tissue was processed for histological analysis as described previously11. 40-μm-thick sections were cut at intervals of 400 μm. Obtained 20–30 slices per pancreas were analysed.
Sections were washed with PBS for 3 × 5 min, permeabilized with 0.2% Triton X-100 (Naxo, Estonia)/PBS solution for 30 min, incubated in 0.5% H2O2/PBS for 1 h, and blocked in 5% donkey serum/1% bovine serum albumin (BSA, Sigma-Aldrich)/PBS for 1 h. Primary and secondary antibodies were diluted in 0.1% Tween-20/1% BSA/PBS. Sections were incubated with mouse anti-insulin antibody (1:800, Cell Signaling Technology Cat# 8138 S RRID:AB_10949314) for 1 h and washed with PBS for 3 × 10 min, followed by incubation with donkey anti-mouse peroxidase conjugated antibody (1:2000, Rockland Cat# 610-703-002 RRID:AB_219700) for 30 min and washed with PBS 3 × 5 min. Sections were incubated in 0.025% diaminobenzidine (Sigma Aldrich)/0.005% H2O2/0.05% CoCl2 (Sigma Aldrich)/PBS for 15 min and washed with water. Dried sections were mounted using PeRtEx (HistoLab) and covered with 0.17 mm coverslip (Deltalab). Images were taken using a Leica SCN 400 slide scanner at 20x magnification. The images obtained were analysed using the ImageJ software. Langerhans islet mass for each animal was estimated by dividing the total islet area by the total pancreas area, and the obtained relative islet area was multiplied by the weight of the pancreas to estimate Langerhans islet mass.
Isolation of islets of Langerhans
Islets of Langerhans were isolated as has been described previously39. In brief, 0.9 mg/ml collagenase (Sigma-Aldrich, #C7657) solution was injected into the common bile duct of euthanized animals; inflated pancreases were collected and tissues enzymatically dispersed. Most of exocrine tissue was removed by gradient separation in Histopaque solution (Sigma-Aldrich). Islets of Langerhans were collected by hand from remaining exocrine tissue under a stereo microscope.
RNA isolation, cDNA synthesis, and gene expression analyses
RNA from islets of Langerhans was isolated using Direct-zol RNA MiniPrep (Zymo Research), according to the manufacturer's protocol. First-strand cDNA was synthesized using random hexamers and SuperScript™ III Reverse Transcriptase (Invitrogen, USA). qPCR using TaqMan Gene Expression Assays, and Taqman Gene Expression Mastermix (Thermo Fisher Scientific) was used to analyse Wfs1 (Rn00582735_m1, binds exon 1 and 2 boundary, therefore also recognizes mutant Wfs1 lacking exon 5), Glp1R (Glucagon-like peptide-1 receptor; Rn00562406_m1), Grp78 (78 kDa glucose-regulated protein; Rn00565250_m1), Atf4 (Activating transcription factor 4; Rn00824644_g1), Bax (Bcl-2-associated X protein; Rn01480161_g1), Caspase1 (Rn01515235_m1), Caspase3 (Rn00563902_m1), Bcl2 (B-cell lymphoma 2; Rn99999125_m1), IP10 (Interferon gamma-induced protein 10; Rn01413889_g1) and Ki67 (Antigen KI-67; Rn01451446_m1) expression. Relative quantification was performed using the 2−∆Ct method, with Hprt1 (Hypoxanthine-guanine phosphoribosyltransferase; Rn01527840_m1) as an internal control. Xbp1 (X-box binding protein 1) splicing was analysed using rat Xbp1-specific PCR as has been described previously40. Integrated density levels were measured using ImageJ software. All gene expression results were normalized to their expression in WT saline-treated rats.
The data are presented as the mean ± SEM and were compared using factorial or repeated measures ANOVA (after Shapiro-Wilks normality test) followed by Tukey post hoc tests. The data were analysed using version 8 of the Statistica software (Statistica, USA). p < 0.05 was considered statistically significant. In some experiments, the sample size is smaller because of tissue limitations preventing a measurement from being performed, not because of the removal of data from the analysis.
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Junker, A. E., Gluud, L., Holst, J. J., Knop, F. K. & Vilsboll, T. Diabetic and nondiabetic patients with nonalcoholic fatty liver disease have an impaired incretin effect and fasting hyperglucagonaemia. J Intern Med 279, 485–493, https://doi.org/10.1111/joim.12462 (2016).
Ishihara, H. et al. Disruption of the WFS1 gene in mice causes progressive beta-cell loss and impaired stimulus-secretion coupling in insulin secretion. Hum Mol Genet 13, 1159–1170, https://doi.org/10.1093/hmg/ddh125 (2004).
Tamura, K. et al. Liraglutide improves pancreatic Beta cell mass and function in alloxan-induced diabetic mice. PLoS ONE 10, e0126003, https://doi.org/10.1371/journal.pone.0126003 (2015).
Ellenbroek, J. H. et al. Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice. Diabetologia 56, 1980–1986, https://doi.org/10.1007/s00125-013-2957-2 (2013).
Wang, P. et al. Diabetes mellitus–advances and challenges in human beta-cell proliferation. Nature reviews. Endocrinology 11, 201–212, https://doi.org/10.1038/nrendo.2015.9 (2015).
Finegood, D. T., Scaglia, L. & Bonner-Weir, S. Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes 44, 249–256 (1995).
Kageyama, H. et al. Morphological analysis of ghrelin and its receptor distribution in the rat pancreas. Regul Pept 126, 67–71, https://doi.org/10.1016/j.regpep.2004.08.031 (2005).
Ehses, J. A. et al. Increased number of islet-associated macrophages in type 2 diabetes. Diabetes 56, 2356–2370, https://doi.org/10.2337/db06-1650 (2007).
McDaniel, M. L., Kwon, G., Hill, J. R., Marshall, C. A. & Corbett, J. A. Cytokines and nitric oxide in islet inflammation and diabetes. Proc Soc Exp Biol Med 211, 24–32 (1996).
Dobrian, A. D. et al. Dipeptidyl peptidase IV inhibitor sitagliptin reduces local inflammation in adipose tissue and in pancreatic islets of obese mice. Am J Physiol Endocrinol Metab 300, E410–421, https://doi.org/10.1152/ajpendo.00463.2010 (2011).
Velmurugan, K. et al. Antiapoptotic actions of exendin-4 against hypoxia and cytokines are augmented by CREB. Endocrinology 153, 1116–1128, https://doi.org/10.1210/en.2011-1895 (2012).
Huang, C., Yuan, L. & Cao, S. Endogenous GLP-1 as a key self-defense molecule against lipotoxicity in pancreatic islets. Int J Mol Med 36, 173–185, https://doi.org/10.3892/ijmm.2015.2207 (2015).
Iglesias, J. et al. PPARbeta/delta affects pancreatic beta cell mass and insulin secretion in mice. J Clin Invest 122, 4105–4117, https://doi.org/10.1172/JCI42127 (2012).
Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R. & Nunemaker, C. S. A practical guide to rodent islet isolation and assessment. Biological procedures online 11, 3–31, https://doi.org/10.1007/s12575-009-9021-0 (2009).
Yusta, B. et al. GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress. Cell Metab 4, 391–406, https://doi.org/10.1016/j.cmet.2006.10.001 (2006).
This research was supported by the European Union through the European Regional Development Fund (Project No. 2014-2020.4.01.15-0012), by grants PUT784 (AT), IUT14064T (MP) and IUT20-41 (EV) from Estonian Research Council.
Maarja Toots and Kadri Seppa contributed equally to this work.
Institute of Biomedicine and Translational Medicine, Department of Physiology, University of Tartu, 19 Ravila Street, Tartu, 50411, Estonia
Kadri Seppa, Toomas Jagomäe, Anton Terasmaa, Mario Plaas & Eero Vasar
Institute of Biomedicine and Translational Medicine, Laboratory Animal Centre, University of Tartu, 14B Ravila Street, Tartu, 50411, Estonia
Maarja Toots, Tuuliki Koppel, Maia Pallase, Indrek Heinla & Mario Plaas
Centre of Excellence for Genomics and Translational Medicine, University of Tartu, Ravila 19, Tartu, 50411, Estonia
Eero Vasar
Maarja Toots
Kadri Seppa
Toomas Jagomäe
Tuuliki Koppel
Maia Pallase
Indrek Heinla
Anton Terasmaa
Mario Plaas
M.P. conceived the study. M.P. and A.T. designed the experiments, M.P. and E.V. directed the study. M.P., K.S., M.T., I.H., M.Pa., T.K., T.J. and A.T. performed animal experiments. M.T. performed biochemical analysis; M.T. and K.S. performed histological analysis. All authors participated in analysis and interpretation of the data and writing of the manuscript. M.T. wrote most of the manuscript. All authors have read and approved the final version of the manuscript.
Correspondence to Mario Plaas.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Toots, M., Seppa, K., Jagomäe, T. et al. Preventive treatment with liraglutide protects against development of glucose intolerance in a rat model of Wolfram syndrome. Sci Rep 8, 10183 (2018). https://doi.org/10.1038/s41598-018-28314-z
DOI: https://doi.org/10.1038/s41598-018-28314-z
Liraglutide, 7,8-DHF and their co-treatment prevents loss of vision and cognitive decline in a Wolfram syndrome rat model
Scientific Reports (2021)
Collaboration for rare diabetes: understanding new treatment options for Wolfram syndrome
Felix Reschke
Julia Rohayem
Thomas Danne
Endocrine (2021)
Rat models of human diseases and related phenotypes: a systematic inventory of the causative genes
Claude Szpirer
Journal of Biomedical Science (2020)
GLP-1 receptor agonist liraglutide has a neuroprotective effect on an aged rat model of Wolfram syndrome
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If you guys take a closer look at my blog now, you'll see that I no longer have Google Adsense ads. This is because someone has click bombed me and Google already replied me saying that they can not reinstate my Adsense account. I'm not here to point fingers or anything, but here to tell you that I will most likely be replacing my old Adsense ads with Amazon Associate ads. I may find another PTC advertising company later. As of right now, the only program I am participating in is the Amazon Associates program.
I will do my best to still come and bring you great content on all my blogs and websites. :) But yeah, not saying you have to or anything. If you buy something from Amazon and you do it through my referral links, I do make some referral money.
Anyways guys, please don't forget to share my blog/website with your friends, family members, co-workers, and everyone else in this world! Thank you!


Transmission of etiologic agents by arthropods is a complex phenomenon, and generalizations are difficult to make. Just because an arthropod feeds on a diseased host does not ensure that it can become infected, nor does it ensure (even if disease agents are ingested) that ingested pathogens can survive and develop. There is considerable misunderstanding about this. When bitten by a tick, people think of Lyme disease (or something similar), often insisting that their physician prescribe an antibiotic prophylactically. Little do they realize that there are many tick species and not all are capable of disease transmission (1). Further, they fail to realize that not every tick in nature (even within a vector species) can be infected. Depending on the disease and area of the country, the presence of an infected tick can be like a needle in a haystack.


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Holiday Inn Resort : Panama City Beach
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Bryan White to Perform at the Holiday Inn Resort August 18!
The Holiday Inn Resort Panama City Beach Guest Exclusive Concert Series gets better every week! Bryan White will join the Concert Series August 18, 2019 by performing on the Escape Deck!
This country superstar debuted his first single "Eugene You Genius" in 1994 at the age of 20 and with that debut under his belt Bryan, would go to become a well-known singer and songwriter.
White has charted seventeen singles on the Billboard country charts, of which four reached number 1: "Someone Else's Star" in 1995, "Rebecca Lynn" and "So Much for Pretending" in 1996, and "Sittin' on Go" in 1997. "So Much for Pretending" was the most successful of these songs, spent two weeks on the Country Music Charts at Number One.
"Between Now and Forever" was certified platinum by the Recording Industry Association of America, and "The Right Place" was certified gold. His fourth album, 1999's "How Lucky I Am", produced 2 top 40 singles, with the song "God Gave Me You "becoming a big hit in the states and by becoming a big hit in the Philippines. The song caught on in the Philippines in 2011 and became something of a phenomenon last year when Maine Mendoza, also known as Yaya Dub, and Alden Richards chose "God Gave Me You" as the theme song for their wildly popular variety show "Eat Bulaga".
Come be a part of the performance by Bryan White on August 18, 2019 at the Holiday Inn Resort! Your room is your ticket so no need to worry about the planning. All you have to do is show up on our Escape Deck, grab a drink from SeaGlass Bar, and enjoy the show while watching the sun go down on the beautiful Gulf of Mexico!
To find our more about Bryan White, our Guest Exclusive Concert Series or book your Gulf-front room today, call our in-house reservations department at 800.633.0266.
Have questions about the area? Just ask!
You can find us on Facebook, contact us here, email us at info@paradisefound.com, or call us Direct at 800.633.0266 and one of our Team Members will be happy to assist you with any questions you may have.
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With a foundation date of 1890, Sevilla Fútbol Club can boast of being one of the two oldest football clubs in Spain. It shares that honor with another Andalusian club, Recreativo de Huelva. Sevilla and Recreativo also made their mark in history by playing the first official football match in Spain; Sevilla came out victorious, defeating their oldest rivals by 2-0. During its long existence, Sevilla has won one La Liga, five Copa del Reys and five UEFA Cups/UEFA Europa Leagues.
The home stadium Ramón Sánchez Pizjuán.
During its first couple of decades, Sevilla FC had to settle for being the best football club in its region. Out of the nineteen Championships of Andalusia played in the period from 1917 to 1940, Sevilla won sixteen, while placing runners-up in the remaining three. They further added to their trophy collection by winning two Copa del Reys in 1935 and 1939.
As it turned out, the two cups were a harbinger of things to come. In 1946, Sevilla won its only La Liga to date, defeating the favored Barcelona by a single point. Another Copa del Rey in 1948 marked the end of their conquests for a long time; even though the club managed to stay near the top of Spanish football for a while yet, a financial crisis in the 70s saw them being relegated to the Segunda Division on numerous occasions during the next three decades.
The new century brought some welcome changes to the club, most notably with Juande Ramos' appointment as head coach. Under his leadership, Sevilla returned to the football map by winning two consecutive UEFA Cups in 2006 and 2007. The latter season also saw them win the Copa del Rey, a feat they repeated in 2010.
After being forced to sell their best players due to another financial crisis, Sevilla shocked everyone by winning three UEFA Europa Leagues in 2014 2015 and 2016. As the UEFA Cup and the UEFA Europa League are considered the same tournament, these latest triumphs saw Sevilla become the most successful club in the history of the competition.
The logo contains a shield with three parts plus a classic soccer ball in the center. The picture in the upper left is the Coat of arms of Seville. In the upper right the initials "SFC" standing for Sevilla Fútbol Club could be seen. The logo has more or less been the same since 1921. Before that, the logo consisted of the initials inside a circle.


By nature most birds are a flock species. Your objective when bonding with your bird is to establish who is the head of the flock. To accomplish this you first must gain the birds trust. NEVER strike the bird, this will only make the bird afraid of you... not trust you.
As with any pet, treat it with love and it will give you love. Treat it unkindly and you will have a mean spirited pet.
Let your bird eat with you. Cockatiels love to eat in a group, so by allowing the bird to eat with you and your family you will gain the birds trust. Most birds will eat just about anything they see you eating so don't hesitate to give it what you are eating. The earlier you introduce new foods to your bird, the better.
No No's for birds are: Chocolate, caffeine, avocado, apple seeds, sunflowers, plug-in air fresheners & candle scents, Reynolds cooking bags, carpet fresh carpet cleaner, Fabreze. These items are deadly to birds.
Teach your bird to step up on command. Also, stick train it. NEVER allow your bird on your shoulder. This gives them the upper hand and they think they are head of the flock.
Always change your bird's water daily and check it's pellet. Change pellet every other day unless it is empty.
Keep your bird's cage clean. However, NEVER use toxic cleaners. Warm soap and water with a little Clorox will do just fine.
Also, never spray aerosols in the same room as your bird. Their lungs are tiny and inhaling the fumes will surely kill them. This includes air fresheners, oven cleaners, ammonia based window cleaners, 409 type cleaners, etc… Also, be careful when cooking with teflon coated cookware. If overheated, it will emit deadly gases that will instantly kill your bird. You get the idea. Safety is the key.
Set a bedtime for your bird. They need at least 8-12 hours daily. Also, never cover your bird unless it is to discipline it, (Screaming). Cockatiels can have "Night frights" and will do bodily harm to themselves, if scared, by thrashing wildly in their cage.
I hope this little information sheet is helpful and I know you will be good birdy parents.


More topics, more Magnus, more support for independent living.
get all the latest life skills adventures.
Magnusmode is working with companies around the world to transform the customer service experience for people with autism and other cognitive special needs.
Our mobile app, MagnusCards®, increases accessibility for your customers with cognitive special needs. In just 4-6 weeks, we deliver custom, branded, digital Card Decks that enable people and their support networks to better access and engage with your brand. Let us work with you to show your customers how to realize maximum value from your products and services.
For people with special needs, the world can be a loud, scary and overwhelming place. The character of Magnus was designed for this community by someone with autism. Magnus helps them to focus and manage activities with decreased stress and greater independence.
Toronto Pearson will become the first airport to participate in the program, setting us apart as leaders in accessibility and demonstrating our commitment to creating a universal experience for all passengers.
We are thrilled by the opportunity to partner with Magnusmode and make our restaurants an inviting space for all our guests.
The Toronto Zoo is delighted to participate in the Magnusmode program, which is a true example of community engagement.
Our solution, MagnusCards®, increases accessibility for your customers with cognitive special needs. Let's get started on realizing your inclusiveness strategy today. Your fully branded Card Decks can be ready to launch in as little as 4-6 weeks.
© Magnusmode 2018. All rights reserved.


Whole North Korea in Tears over loss of great Leader Kim Jong Il: Tears in their eyes and anguish plain on their faces, these videos show the people of North Korea in mourning at the sudden death of leader Kim Jong Il.
The footage, taken in the capital Pyongyang, shows hundreds of people weeping and punching the ground in despair in front of a memorial to the 69-year-old dictator.
School children, factory workers and armed service personnel can be seen crying in front of images of the diminutive leader.


The waters of Lake Müritz stretch out before you, and grand castles jut from forested slopes.
You think you're in paradise as you guide the boat to the next resort, or as you nose into a new waterfront town with its very own Baroque palace.
The big lake is all part of a Müritz Short Break river cruise in Germany!
The waters of the Havel River flow past princely Potsdam, a UNESCO World Heritage site and often called Berlin's Little Sister.
As you lounge amongst friends with cocktails on the sundeck, you can scarcely believe you're here!
Brandenburg and Mecklenburg have lured you for city nightlife and the natural beauty of river cruises in Germany's spectacular nature reserves.
The Spreewald Nature Reserve covers approximately 480 square miles and has more than 300 canals lacing low marshes that are home to myriad species of wildlife.
This is your wondrous playground for a Germany river cruise! It's not so far from the discos, upscale restaurants, museums and historic sites in the cities, but you feel like it's a whole world away!
Nearly a third of Brandenburg is designated nature reserves.
In fact, 15 protected areas teem with wildlife in marshes and forests, and the region is dotted with more than 3,000 lakes studded with mediaeval castles and towns! You've chosen an ideal locale for Brandenburg river cruises in Germany!
The former centre of the Duchy of Prussia is endowed with exquisite country charm.
Palaces and châteaux imbue Brandenburg and Mecklenburg with an ambiance of elegance.
The incredible culture and beauty of Germany river cruises is obvious as you stroll in mediaeval market squares and browse in the shops in cities like Mirow and Rheinsberg .
Pampering yourself is part of the fun, so you plan to thoroughly enjoy the watersports and resort amenities at Marina Wolfsbruch !
A Berlin river cruise in Germany is a highlight of your boating holiday, so it's no surprise that you want to see a major city of the world.
Go by boat or take advantage of public transportation and ride in from Potsdam to sightsee .
Berlin boasts more than 150 museums and historic sites, upscale shops, fine dining and lively nightlife.


Bring in the New Year with a new dive destination!
Utila is a Bay Island off the coast of mainland Honduras in Central America. With warm waters year round, it is a migratory destination for the largest fish in the sea—the whale shark! Dive sites also include the famous Halliburton wreck, sloping ledges, walls, and creature-filled sandy areas off the beautiful healthy reefs.
Utila Lodge is the largest over-the-Water dive resort on the island, with eight beautiful bungalows each equipped with a queen bed, double bed, AC, WiFi/Cable TV, private patio and hammocks.
Does not include airfare, gratuities, gear, alcohol, or passports/visas.
We are happy to help arrange transportation and land based tours.
Contact Jenn with any questions!


While Kanye's away, ranting about "Suit & Tie, his baby mama will play. Kim Kardashian found herself among Hollywood royalty last night, as well as all other L.A. denizens without a ticket to the actual awards portion of Oscar Sunday. But who cares! The mom-to-be, her sister Kourtney, and pal Nicki Minaj were just happy to have a reason to party. Keeping dreaming of one day achieving marginal stardom, kids. It might be just enough to get you into Elton John's annual award show fete. Click through for a look at Selena Gomez, Heidi Klum, Miley Cyrus, and the Oscar faves who celebrated well into the night.
Because after the show it's the after-party, and after the party it's… time for bed? Never.
Of course, Nicki and the elder Kardashians sisters weren't the only ones reaping the benefits of their fame for couture formal wear and an open bar. The friends mingled with a crowd made up of Oscar winners, reality stars, and locals hunting for some free appetizers, gaining important style perspective along the way.
Oscar night fashion! This is what it's all about.
We're assuming it was a long night, but at least things ended well.


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Home » FAME » television » Neil Patrick Harris is a Convincing Count Olaf
Neil Patrick Harris is a Convincing Count Olaf
Netflix had recently announced that Neil Patrick Harris would be playing Count Olaf in their upcoming series, A Series of Unfortunate Events. Some sources in Vancouver caught photos of the series' antagonist looking as frightening as the character in the book, and we approve. We look forward to seeing how Neil Patrick Haris' and Jim Carrey's Count Olafs differ, and where this series will go.
The series will be 13 episodes, however, a release date has not been announced yet. Since filming is already underway, it is a safe bet that we will be seeing a debut in late 2016 or early 2017.
We have included some photos of Neil Patrick Harris as Count Olaf below. Enjoy!


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BUMPER BESTIES
How six pals pregnant at the same time are supporting each other through the trials of motherhood
Julia Etherington
MATERNITY leave can be a lonely time for new mums.
Sleepless nights, endless breastfeeding and cartoons on repeat can mean daily adult conversation becomes a distant memory.
These six best friends were all pregnant at the same timeCredit: Matthew Pover
That is why having a "maternity buddy" can be a lifeline for many women — a close friend who is pregnant at the same time so they can share parenting highs and lows.
So when Beth Croft announced her pregnancy — and five of her friends followed suit — it was a baby bonanza that none of them could have expected.
Now the super six from Teesside, who have been best pals for decades, are readying themselves for maternity leave and joke that there must have been something in the drinking water locally.
Primary school teacher Beth, 34, is married to Jonny, also 34, and was the first to announce she was expecting.
Beth was the first to announce her pregnancy and has already had her baby boy ParkerCredit: Matthew Pover
The couple already have daughter Ivy, who is six, and Beth gave birth to son Parker on May 23.
Next to reveal she had a bun in the oven was social care worker Lucy Mallam, 34.
Beth says: "After I announced my pregnancy and then Lucy announced hers, I couldn't believe it. And as the others did, too, I was gobsmacked.
"When I had Ivy it was a little bit different. It was just me, really, and I had a traumatic birth with her.
Lucy was the next to announce her pregnancy and is due on July 24Credit: Matthew Pover
"She was six weeks premature and I had four blood transfusions after haemorrhaging. She was 4lb 7oz and was in the neonatal unit for a few days.
"She was breathing on her own and, thankfully, soon thrived. But it was quite a difficult time.
"Parker was born by C section at full term, weighing 6lb 4oz. He's such a placid baby and really relaxed and easy.
"Now I'm just waiting for the others to start their maternity leave. I'm planning to take full advantage of having the girls round and meeting up lots. I can't wait."
Naomi is also pregnant with her first childCredit: Matthew Pover
Lucy, who is now 35 weeks pregnant, is always on hand to offer advice to the others, as this is her fourth child with husband Aaron, 35, a prison maintenance worker.
She says: "When I had my first child, it was years before any of my friends began starting families. Maternity leave was lonely and I felt so clueless, being just 21."
This time, she has the support of her pals who will also be able to muck in with parenting duties with her other children, Harrison, 12, Frankie, eight and Jude, four.
Lucy, who is expecting a girl on July 24, continues: "It was like another person was announcing they were also pregnant almost every month.
"It became a running joke about who would be next."
Emma waited until she was 12 weeks pregnant to announce her newsCredit: Matthew Pover
Sure enough, the next announcement arrived around three weeks after Lucy's, when pal Naomi Lee revealed she was expecting her first child, on August 11.
Naomi, 36, who works for a construction company, says: "It's been such a relief and, at times, a godsend to be able to ask the girls questions and not feel embarrassed about my lack of baby knowledge.
"Luckily pregnancy has been a breeze. I haven't had sickness, although I do get swollen cankles at the end of the day.
"It's nice to know you're not alone in whatever you're feeling. We've swapped thoughts and chatted about everything from nursery ideas, personal experiences and even shared maternity clothes."
Laura Hatfield is 24 weeks pregnantCredit: Matthew Pover
Naomi is even more thankful for her pregnant buddies as her partner Matthew Allison, 37, is an oil company worker.
She says: "I'll be doing a lot of it on my own as Matthew works away a lot, which can be lonely. So I'm lucky to have our group's support."
Fortunately, all the ladies have experienced healthy pregnancies, although one, Emma Harding, was a little more apprehensive at the beginning. The finance team leader, married to salesman Phil, 34, waited until her 12-week scan before revealing her news.
She says: "I'd had some issues at the start with bleeding and didn't want to take the sunshine and excitement away from the others if anything went wrong.
Lyndsey is the last one due as she is just 18 weeksCredit: Matthew Pover
"Luckily everything is OK and apart from being extra tired, the pregnancy's been great."
Already mum to four-year-old William, Emma, 33, is expecting another boy on September 12.
She says: "We already had a big group chat for our wider circle of close friends, but with so many of us falling pregnant, we decided not to bore them with baby talk so we set up another group.
"We bombard each other every hour. Even if it's the middle of the night, someone will probably be awake, too.
The women have known each other from secondary schoolCredit: Matthew Pover
"Our conversations can vary from moaning about no sleep to how our sex lives are going — or not, ideas about decorating our nurseries and comparing symptoms. It can get quite graphic, but if you can't share with your girls, who can you share with?"
And Laura Hatfield, 24 weeks pregnant, couldn't agree more.
Already mum to Oliver, ten, from a previous relationship, she reckons her maternity leave this time around is certain to be much more fun.
Bank worker Laura, 34, says: "The first maternity leave I had was lonely as most of the girls were out partying and not thinking of starting families. I spent most of it alone or with Mum.
The women have had lots of baby showers and announcements between them this yearCredit: Matthew Pover
"I've known most of the girls since secondary school and we went through college together. So to be pregnant at the same time, too, has meant a ready-made support network.
"We tend to ask each other about the more grim side of pregnancy, like constipation, wondering whether our bumps are too big or small, or how to get shoes on fat, swollen feet.
"We share monthly photos of our bumps to compare size."
Laura, who is expecting with partner John Patino, 32, also a bank worker, says: "Every month we're either having a baby shower or welcoming a new arrival, so it's pretty busy.
Gemma Atkinson says she's been 'vomiting all night' after FOURTH water infection and hospital dash during pregnancy
"We talked about having joint baby showers, but decided to still have individual ones to keep it special for each person. It'll be great seeing the babies grow up together and be friends, too."
The final announcement came from Lyndsey Thompson, who works for a housing association.
Now 18 weeks pregnant, she is expecting her first baby on November 30 with partner Marc Price, 32, a maintenance worker.
She says: "I was the last one to announce I was pregnant, but we're still looking at our wider group and wondering who will be next. It's a really exciting time — I just think it's crazy I've got this little person inside me." Lyndsey, 34, adds: "Having friends all in the same boat is great.
I'm clueless about babies so it means I don't have to bombard my other friends who aren't pregnant with questions that would be boring for them.
"And from a practical point of view, I'm already being offered hand-me-down baby clothes."
The ladies' partners all get on well when the group hang out together, but admit they have been suspicious about these conveniently well-timed pregnancies.
Lucy says: "All of our husbands and partners have joked about us girls planning this together so we can all have some time off with each other.
"You'd think we had, but we definitely didn't."
A mum has revealed how she was bullied for the size of her pregnancy bump
MEAT AND GREET
Mum doesn't feed her son, two, ANY veg but he eats raw brains for dinner


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NYC, 1970
Elton John, Dodger Stadium, Ready at the Bat, Los Angeles, CA, 1975
Elton John, Dodger Stadium, Los Angeles, CA, 1975
Elton John Feathers
Elton John, Dodger Stadium, Los Angeles, CA, 1975, B&W
Elton John, Nudie Suit, London, 1972
Elton John, Dodger Stadium, Back Turned, Los Angeles, CA 1975
Elton John, Sundown Theatre, Edmonton, North London, 1973
Elton John, NYC, 1971
David Cassidy and Elton John, New Zealand, 1974
Elton John & Stevie Wonder, Starship Airplane 1973
Elton John, Windsor, England, 1974
Elton John, New York City, 1980
Janis Joplin, Newport Folk Festival 1968
Patti Smith, Eric Anderson
Miles Davis, Tanglewood Music Festival, Lenox, MA 1970
Joni Mitchell & Leonard Cohen, Newport Folk Festival, 1967
Bruce Springsteen Wild and Innocent
Bruce Springsteen & the E Street Band