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Lupine Publishers| The Effect of Glibenclamide Administration on Gastrin Release in Diabetic Patients

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

The effects of sulfonylureas on gastrointestinal function in man is not yet quite clear. The aim of this study was to investigate the effect of oral administration of glibenclamide on gastrin release in patients with non-insulin dependent diabetes mellitus. Twelve non-insulin dependent diabetic patients (six men, six women, median age 57 years, range 46-63 years) were studied. Glibenclamide or placebo were given on different days and in a random order 10 minutes before a standard meal (73.6 g corned beef + 5ml olive oil + 60g bread). Blood samples for the determination of gastrin, glucose and C-peptide in serum, before (-15 and 0 minutes) and 30, 60 , 90 , 120 and 180 minutes after the standard meal, were obtained. Initial mean values of gastrin in serum did not differ significantly between the two meals. As expected gastrin levels increased significantly after taking the meals. However, no significant differences concerning mean gastrin concentrations between the two meals were noted at all time intervals studied, although there was a trend for the glibenclamide-preceded meal to exert lower gastrin values, especially at 60 minutes (p: 0,06). Mean serum glucose levels were, as anticipated, significantly lower after the glibenclamide-meal. Similarly, serum C-peptide concentrations were higher after this meal. It is concluded that acute glibenclamide oral administration does not influence post-stimulatory 3 gastrin levels in non-insulin dependent diabetic patients. Thus, in the clinical situation, sulfonylurea administration does not seem to interfere with gastrin release.

Introduction

The effect of PPIs (Proton Pump Inhibitors) on serum gastrin levels has been well known since the early years of patient treatment with omeprazole [1]. On the contrary, the effects of sulfonylureas on gastrointestinal function in man is not yet quite clear. Sulfonylureas are known to have various extrapancreatic actions [2]. In addition to their stimulatory effect on insulin release from pancreatic islets. However, the effects of sulfonylureas on gastrointestinal function and gut hormones release remain unclear. More specifically, as it regards gastrin, there are only a few studies in which the effect of sulfonylureas is investigated [3,4]. furthermore, these studies show a discrepancy between results obtained in man and animal models [5,6] and, in addition they describe only the effect of sulfonylureas on fasting and not the postprandial serum gastrin concentrations [3,4]. The present study was undertaken, therefore, to investigate the effect of glibenclamide oral administration in patients with Non-Insulin Dependent Diabetes Mellitus (NIDDM) in combination with a test meal.

Results

Initial mean values of gastrin in serum did not differ significantly between the two meals and were within the normal range (<90 pmol.1). As expected, gastrin levels increased significantly after taking the meals (Table 1). However, no significant gastrin concentrations between the two meals (meal + glibenclamide and meal + placebo) at all intervals studied, although there was a trend for the glibenclamide-preceed meal to exert lower gastrin values, especially at 60 minutes (p:0.063) (Table 1). Blood glucose variations are summarized in (Table 2). Mean serum glucose levels were, as anticipated significantly lower after the glibenclamide-meal as compared to those after placebo-meal. Similarly, postprandial C-peptide concentrations in serum were significantly higher after the glibenclamide-meal comparing to the placebo-meal (Table 3).

Table 1: Mean serum gastrin values ±SD (pmol/l).

Table 2: Mean serum glucose levels ± SD (mg/dl).

Table 3: Mean serum C-peptide concentrations ± SD (ng/dl).

Discussion

In this study, gastrin concentrations in serum were found not to be altered significantly in NIDD patients after oral administration of glibenclamide, in combination with a meal, as compared to those who received the same meal with placebo. Although there was a trend for patients taking glibenclamide to present lower postprandial serum gastrin levels, this difference was not statistically significant. These findings are in agreement with the results of a previous study in which injectable solution of glibenclamide was administered intravenously or per os to healthy volunteers [3]. In the above study, gastrin levels were estimated in periphal and portal blood and were found to be essentially unchanged, under all conditions studies [3]. In another study, tolbutamide was reported to inhibit gastrin release in man [4]. In that case the drug was administrated intravenously or per so to normal subjects as well as in patients with atrophic gastritis, duodenal ulcer and IDDM [4].

Our study Differs from the previous reports:

a) Because it concerned exclusively patients with NIDDM, who are mainly treated with glibenclamide and

b) In the parallel administration of a test meal.

Therefore, it is obvious that the present study was planned accordingly to simulate the everyday conditions in diabetic patients taking a meal in combination with glibenclamide. These results, as well as the above-mentioned study [4], suggest a possible inhibition of gastrin release by Sulfonylureas in man. However, in animal models, gastrin release has been reported to be stimulated by sulfonylureas [5,6]. Indeed tolbutamide was found to stimulate both somatostatin and gastrin secretion from the isolated perfused rat stomach [5]. Also, glibenclamide was reported to stimulate gastrin release from the antral mucosa of cats [6]. It is possible that differences in animal species, as well as in the experimental design and drug dosage may account for this discrepancy. Basal gastrin values of diabetic patients in the present study were in normal range. Hupergastrinaemia has not been reported previously in diabetic humas, except in the diabetic pesuedo-Zolinger-Ellison syndrome [7] and a number of patients with clinical manifestations of autonomic neuropathy [8-10]. It has been further suggested that increased serum gastrin leels in those NIDD patients are not related to hypochlorhydria but, instead, are resulting from the autonomic dysfunction [8,9]. In the present study, patients did not show clinical manifestations of autonomic neuropathy. This, as well as the fact that they did not have impaired renal function, might well explain the normal initial mean gastrin values. Finally, it is noted that the present experiments deal with acute glibenclamide administration. Also, it would be interesting to investigate further the possible effect of sulfonylureas on gastrin levels in diabetic patients presenting hypergastrinaemia.

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Lupine Publishers| An Evidence-Based Herbal Supplement for The Control of Metabolic Syndrome

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

Background: Metabolic Syndrome (MS): Overweight, Obesity, Hypertension, Hyperglycemia and Hypercholesterolemia, are generally accepted today as clinical signals leading to cardiovascular diseases. Control of MS is therefore of a common health concern. While drug treatment is yet not available or may not be creditable, developing an effective health supplement against MS is highly justified.

Methods: A herbal formula composed of four herbs known to have anti MS pathological effects was used for in vivo and in vitro biological researches to verify its pharmacological effect, and subsequent pilot clinical trial.

Results: In vitro study: Adipocyte viability and cholesterol uptake, liver cell viability and anti-glycaemia effects, all gave positive results of good control. In vivo study testing herbal formula’s effects on obese mice also showed very promising results. In clinical trial, measurements of body weight, body circumferences, BMI, as well as liver fibrosis, all showed good responses after the herbal formula consumption.

Conclusion: Our efforts on the creation of an Evidence-Based Specific Supplement for the control of Metabolic Syndrome have harvested highly positive data in the laboratory. The subsequent 3 months’ pilot clinical trial showed positive data on the control of blood lipids, general body measurements and liver steatosis.

Keywords: Gene Expression; Lipid Droplets; Mitochondria; RNA Sequencing; Type 2 Diabetes Mellitus

Introduction

As cardiovascular diseases have become dominant causes of mortality, other related pathological presentations are gaining public attention [1,2]. Obesity is the commonest observable indicative of “unhealthiness”, leading to cardiovascular problems. Thus, simple objective measurements of obesity like Body Mass Index (BMI), blood lipids and cholesterols are also gaining much public concern [3]. Blood pressure and blood sugar levels naturally fall into the same checklist of safety requirements which are mainly affecting cardiovascular health [4].
As time goes on, the five major influences on cardiovascular health have thus been put together as the “Metabolic Syndrome” (MS): overweight, obesity, hypertension, hyperglycemia and hypercholesterolemia [5]. Another associated pathology, viz. fatty liver, turns up, as many sufferers of MS are found to have different degrees of liver dysfunction and fat deposits gradually leading the way to liver fibrosis [6].
For decades, varieties of interventions are available for the control of the different aspects of MS, particularly in areas that require specific effective control like hypertension and hyperglycemia. It is realized now that a better way to deal with the problem is to broaden the area of concern so that MS can be taken together in a multi-facetted preventive endeavor. Although specific single target pharmaceuticals are available for solitary treatment, a more ideal alternative could be some extra-pharmaceutical multiple target, harmonizing supplement to take care of all components of the MS [7,8].

Medicinal Herbs

A number of Medicinal herbs have been traditionally used as supplement for patients with diabetes mellitus and obesity. They are chosen for our study on metabolic syndrome. Platform studies would include bioactivities related to cardiovascular problems [9,10].
We selected four edible herbals items for the Chinese pharmacopeia to be tested. They are: (Table 1).

Table 1: Four edible herbals items for the Chinese pharmacopeia to be tested.

The combined formula (2MSC) would be tested for the control of body fat and sugar metabolism, while its effects on blood pressure, blood lipids and liver function would carefully be studied [17]. The design of this study on MS has chosen an emphasis on the effects on fatty liver [18].

Methods

Preclinical Studies

In vitro experiments included the following:

To investigate specific bioactivities of individual herbs and the combined formula

I.Testing the effects of Crataegus Fructus and the formula on the adipocyte viability and cholesterol uptake using adipocyte and cacocell cultures [19].

II.Testing the effects of Schisandra Fructus and Silymarim Marianum separately and the combined formula on liver cell viability using HepG2 cells [20].

III.Testing the antiglycemic effects of Momordica and the combined formula using Brush Border Membrance Vesicle model via its glucose uptake obstruction [21-23].

In vivo Experiments

Male C578 1/6 mice were use. Obesity induction was achieved using forced high-fat feeding for 8 weeks. The obese mice were treated with normal diet and continual high fat diet with 2% and 4% combined herbal formula [18]. At the end of 8 weeks for the low dose and 12 weeks for the high dose, the animals were sacrificed to have comprehensive checks on Body Weight, Blood examinations for lipid assessments and liver examinations.

Results of Laboratory Studies

In Vitro Studies

3T3-L1 preadipocytes differentiation cell assay showed the effects of the different concentrations of 125 μg/ml, it was a dose that induced significant toxicity to cells, and there was no dose– response effect observed [19].
Fluorescent tagged cholesterol-treated Caco-2 cell assay

The effect of the different concentrations of Crataegus Fructus aqueous extract and the herbal formula extract on cholesterol uptake in Caco-2 cells. Crataegus Fructus aqueous extract significantly prevented the cholesterol uptake in Caco-2 cells in a dose dependent manner. Herbal formula extract on the other hand had no significant effect on the cholesterol uptake in Caco-2 cells [19].

Animal Studies

The body weight, adipose tissues weight and liver weight were measured. In the 8-week treatment study, the High-fat diet (HF-fed) animals significantly gained more weight than chow-fed animals. Among all HF-fed animals, there was a trend for a reduction in body weight of 4% among herbal formula fed animals, starting from week 9 onwards. Three types of adipose tissues (epididymal fat, peri-renal fat, and inguinal subcutaneous fat) were isolated and weighed. High-fat diet induced obesity in mice compared to normal chow-fed mice after 16 weeks and 20 weeks, as evidenced by the significant increase in all three types of fat pad mass to body weight ratio: epididymal fat pad (p < 0.01 for both 8-week and 12-week treatment studies), inguinal fat pad (p < 0.01 for both 8-week and 12-week treatment studies), and perirenal fat pad (p < 0.001 for both 8-week and 12-week treatment studies).
Livers were isolated, weighed and presented as liver to body weight ratio. High-fat diet induced an increase in liver to body weight ratio in both 8-week and 12-week treatment studies [19]. 2% and 4% herbal extracts dose-dependently reduced the liver to body weight ratio in both treatment periods.

Liver histopathology and inflammation assessment

Livers from mice fed on different diets were analyzed histologically. Normal-chow-fed animals demonstrated the histological sections of normal livers. In contrast, H&E sections from HF animals revealed the presence of a large number of circular lipid droplets within the hepatocytes. These lipid inclusions were clearly reduced in size as well as number in the livers of all herbal formula treated animals.

A Pilot Clinical Trial

Title: Pilot Clinical Study to evaluate the effects of the innovative herbal formula 2MSC in subjects with Metabolic Syndrome.

Hypothesis: 2 MSC is effective for the Management of MS, with particular emphasis on liver fibrosis (Fatty liver).

Study Design

An open label pilot study conducted with 30 overweight adult men and women were assigned to take 2 MSC daily for 3 months.

Study Population

Subjects recruited were 40-66 years of age, with BMI between ≥25kg/m2 and ≤37.7kg/m2. They agreed to attend all study visits and to keep their normal dietary habits and usual physical activities. Subjects were excluded if they were diabetic (on diabetic medication for more than four weeks); on cardiac and statin related drugs; on immune-suppressive drugs. Cigarette smokers, pregnant or lactating women were excluded. A total of 30 volunteers with no past history of allergy to herbal medicine were recruited.

Study Procedures

The study started after signing the proper consent. Duration of treatment lasted 12 weeks. Monthly phone calls were conducted to monitor the progress, compliance and adverse effects. Volunteers were reminded to keep their usual dietary habits and physical activities.

Data Collections

Demographic and basic measurements related to MS including Body Weight, BMI; Waist Circumference; Hip Circumference and Neck Circumference. Blood testing included fasting blood sugar, Liver function tests, Renal function tests. Other items related to Lipid metabolism included total cholesterol (TC), Triglycerides (TG), Low density (LDL) and high-density lipoprotein cholesterol (HDL), and non-high-density lipoprotein cholesterol. Adiponectin and some immune cytokines were also taken. Fibroscan of the liver was done at baseline and final visit. The Quality of life was checked with the standard SF-36 scoring sheet.

Primary Outcome

The primary outcome included the decline of Body Weight, Waist Circumference and Lowing of blood triglyceride TG.

Statistical Analysis

Excel 2016 (Microsoft Corp, Redmond WA) was used for data entry. Statistical analysis was performed using SPSS Base System ver. 25 (IBM SPSS Inc., Chicago IL.). Statistical analysis (descriptive statistics and Student t tests) was performed using SPSS ver. 22 (IBM SPSS Inc., Chicago IL.). Paired t-test was utilized to evaluate the difference between pretreatment mean and post-treatment mean. The percent changes in CAP Reading from baseline to 12 weeks of treatment with 2MSC were analyzed by using Chi-Square test.

Results

Compliance was excellent. No subject withdrew during study period. Adverse effects reported were all mild, including loose stools and mild abdominal discomfort. Liver and kidney functions remained normal. Bodily measurements all showed clear tendencies of improvement with convincing p values (Table 2).

Table 2: Changes of physical measurements of the volunteers.

BW: Body Weight; BMI: body mass index.

The blood checking data indicative of lipid metabolism also showed very positive decline in the triglycerides (Table 3). Fibroscan study showing the “rigidity” of the liver through the anterior abdominal wall, demonstrated softening from 315.3 (49.7) to 291.0 (44.1) p=0.006 (Table 3).
Results of fibroscan for fatty liver study gave an overall positive effects of the herbal formula slowing down the progress of liver fibrosis when the controlled attenuation parameter (CAP) data revealed by the fibroscan were analyzed (Figure 1).

Table 3: Changes in the lipid profile and Fibroscan after administration of 2MSC.

TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; * 12-week minus Baseline.

Figure 1: Overall Fibroscan Results: Comparission of controlled attenuation parameter (CAP) values.

Figure 2: Comparison of CAP Reading.

When the liver conditions of the volunteers were classified into four different groups of liver steatosis, as: minimal, mild, moderate and severe, it was interesting to find that the herbal formula influenced only the moderate group significantly, while the minimal to mild group and severe group were unaffected. (Figure 2).

Discussion

Functional foods or nutraceuticals which have potential anti-obesity properties have attracted great attention. Schisandrae Fructus is a Chinese herb traditionally used as a liver tonic. Silymarin, an extract of the milk thistle (Silybum marianum), is a dietary supplement that is widely used in Europe for the prevention and treatment of liver problems. Crataegus Fructus (hawthorn) is traditionally used to promote digestion and dissipate food stagnation. Momordica charantia (bitter melon) is traditionally used for the treatment of diabetes in Ayurvedic Medicine. Our in vitro results suggested Crataegus Fructus aqueous extract exerted potent inhibitory effects on 3T3-L1 preadipocytes differentiation and cholesterol uptake into Caco-2 cells. Schisandrae Fructus aqueous extract and milk thistle exerted inhibitory effects on oleic acid-induced fatty liver in HepG2 cells. Momordica charantia extract on the other hand, exerted significant inhibitory effects on the glucose uptake into BBMV. Our in vivo results showed that our herbal formula exhibited a trend to reduce diet-induced increase in body weight and fat pad mass (epididymal, perirenal and inguinal fat). It also significantly reduced diet-induced increase in liver weight, liver lipid, and plasma lipid dose-dependently. High-fat diet induced a significant reduction in adiponectin level which was significantly improved by herbal formula supplementation at 4%. The herbal formula also significantly reduced diet-induced inflammation in the liver at both doses.

Metabolic Syndrome is currently understood as a combination of commonly co-existing symptoms some of them clearly present uniquely as an outstanding disease, like diabetes mellitus and hypertension, while others present as potentially threatening pathologies. Although adequate treatment for diabetes and hypertension are available, there is a common wish that they could be controlled at an early stage, so that progression could be curbed [23].
Apart from a rigidly followed lifestyle recommended for the cardiovascular system and obesity, which is not easy for the general public, food supplements have been used to help [24,25]. Our pilot clinical study ventured to develop an evidence-based supplement specific for MS, adopting a broad view covering all five problems: obesity, body weight, BMI, blood pressure, and blood lipids. Changes in liver fat and adiponectin were also explored [26,27]. Our pilot clinical study showed an overall body weight loss from 71.4 to 70.6 kg (p=0.027) and a BMI 28.0 to 27.7 (p=0.053) within a short period of 12 weeks.
The body circumference (external fat collection) shrinkage as detected through waist and hip also showed significant decline (p=0.014 and 0.011).
Since neck circumference measurement has recently been endorsed as a simple practical assessment for body fat, our neck measurements were supportive to the innovation and showed a 37.0 to 36.0 cm decline [28,29].
Useful results were provided in the blood tests. Liver function and renal function tests stayed normal, indicating the safety of the formula. With regard to the lipids: there were declines in triglycerages (p=0.044) and an increase in high density lipid (p=0.034) [30]. Fasting blood sugar remained stable.
The overall results were promising but the trial period lasted only 12 weeks and the number of volunteers was small. More than 50 Traditional Chinese Medicine formulae have been used to treat MS, basing on classical theory “Kidney Health”, cardiovascular support, and general harmonizing effects [31]. The 4-herbs selected to form the 2MSC formula were taken from classical recommendations subsequently scrutinized on bioactivity platform, to provide pharmacological evidence.
Liver scanning using Ultrasonic Fibroscan device is a simple qualitative and quantitative way to evaluate fat contents in the liver suspected of fatty changes. In our study, liver tissue resistance to Ultrasonic pressure was found to decline from 315.3 to 291.3 (p=0.010) within a period of only 12 weeks (Figure 1) (32-35). There are many reports on the study of non-alcoholic fatty liver in response to supplements [25,36-39] mainly targeting on the liver pathology alone. Our study took a broader view: other factors leading to MS should also be contributing towards fatty changes in the liver.
Since the discovery of Adiponectin as a factor very much affecting the Metabolism of fatty tissues responsible for the metabolic cycle connecting fat deposit and carbohydrate consumption, the behavior of adiponectin is often included in studies related to MS [40-43]. In our preclinical animal experimental study lasting 12 weeks, we found a high dose of the combined formula gave an increase of adiponectin suggesting that carbohydrates metabolism could have been promoted [19]. In our pilot clinical trial, however adiponectin was found to be either raised, remained stable or decreased. This might be due to the low dose effect or that the study lasting only 12 weeks. Using the SF36 questionnaire for the volunteers’ selfevaluation of their physical, mental and social well-being’s did not give remarkable results.

Conclusion

Up to today there is no FDA approved medications for the treatment of non-alcoholic fatty liver disease (NAFLD). The American Association for the Study of Liver Diseases (AASLD) suggested the combination use of vitamin E (an antioxidant) and pioglitazone may be helpful but not all patients would benefit from it. For patients diagnosed with NAFLD, the first line of treatment usually involves weight loss through a combination of a healthy diet and exercise. According to the AASLD guidelines, it was recommended that 10% body weight loss would lead to improvement of the steatosis and inflammation related. Previous studies also found that lifestyle modification could significantly improve the mean fibroscan CAP value (278.57±49.13 dB/m VS 252.91±62.02 dB/m, p=0.03). Thus 6 months of lifestyle modification which include moderate intensity physical exercise 3 days per week each for 45 minutes; plus a restricted caloric intake of 25-30 kcals/kg/day could better protect liver health after tremendous individual efforts and This obviously involves hard work and determination.

Our efforts on the creation of an Evidence-Based Specific Supplement for the control of Metabolic Syndrome have harvested highly positive data in the laboratory and in the subsequent 3 months’ pilot clinical trial. Encouraging results were obtained in the control of blood lipids, general body measurements and liver fibrosis. It is envisaged that coupled with more exercises and dietary control, better results could be expected. Further clinical studies would be very much warranted.

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Lupine Publishers| Diabetes in Older People: Comprehensive Approach

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

The clinical management of older people with diabetes requires a comprehensive evaluation and a holistic approach for the individualization of objectives and strategies of treatment. In older people with diabetes, geriatric syndromes, frailty and sarcopenia are considered at present as a third category of chronic complications. These situations are added to traditional microvascular and macrovascular complications, leading to significant disability and increasing the costs. In this context, two clinical scenarios can be considered: the first one, elderly subjects without significant comorbidities and good functional condition, in which an approach to diabetes similar to that of younger patients must be made. The second scenario, elderly and frail subjects, in which it will be essential a correct identification of these conditions and the evaluation of geriatric syndromes. This evaluation will guide the adaptation in the goals of treatment and in global management of diabetes.
Some basic principles should guide our decision-making: starting drugs at low - medium doses, with progressive increase according to tolerability; selection of drugs according to evidence-based medicine (considering the limited evidence in this age group), favoring agents with the lowest risk of hypoglycemia, avoid polypharmacy. Finally, patient´s safety and quality of life should be the main objectives.

Keywords: Diabetes; Older; Frailty; Evidence-Based-Medicine

Opinion

Clinical management of older diabetes people requires a comprehensive evaluation and a holistic approach for the individualization of objectives and strategies of treatment. Geriatric syndromes, frailty and sarcopenia are considered at present as a third category of chronic complications [1]. These situations are added to traditional microvascular and macrovascular complications, leading to significant disability and a significant increase in costs.
In this context, two clinical scenarios can be considered: first, elderly subjects without significant comorbidities and without frailty, in which an approach to diabetes similar to that of younger patients must be made. The second scenario, elderly and frail subjects, in which a correct identification of frailty and an evaluation of geriatric syndromes is mandatory, guiding modifications in the goals of treatment and in the global management of diabetes.

Initial Approach

1. Consider evaluation of medical, functional (self-care skills) and geriatric sphere to establish a frame of reference that determines the objectives and therapeutic strategies diabetes management (Evidence B) [2].
2. Assess presence of geriatric syndromes (polypharmacy, cognitive impairment, depression, urinary incontinence, falls, chronic pain) as conditions that interfere with patient’s management of diabetes and reduce quality of life (Evidence B) [2].

Figure 1: Comprehensive approach in older people with T2DM.
Bold therapy: grade A evidence. * Clinical situation: Intermediate / complex HbA1c 7-8%, TA <140/90 mmHg; very complex HbA1c <8.5%, TA <150/90 mm Hg.
ASA, acetylsalicylic acid; BP, blood pressure; LDLc, LDL cholesterol; y., every “number” years; eGF, estimated glomerular filtration; ACR, urine albumin creatinine ratio; HF, heart failure (evidence limited to patients at risk of heart failure or patients with FH diagnosis and reduced ejection fraction); Ŧ eGFR <30 mL/min/1.73 m2: Initiation not recommended, but once established, it can be maintained until the start of dialysis.
GLP1ra, glucagon-like peptide-1 receptor agonists; SGLT2i, sodium-glucose transport protein 2 inhibitors; DPP4i, dipeptidyl peptidase 4 inhibitors; Glarg, glargine

3. Evaluation of frailty. The most validated and simple evaluation tools are Fried criteria and FRAIL scale. Consider potentially reversible causes that contribute to frailty such as presence of hypothyroidism, vitamin D deficiency, anemia, etc., is advised [3].
4. In those over 65 years of age, an early diagnosis of mild cognitive alterations is recommended, at diagnosis and subsequently annually [2]. Pfeiffer questionnaire or Minimental test are validated tools. In patients with cognitive dysfunction, simplify treatment, and adapt care structure.
5. Patient safety, preferences and quality of life should be the main objectives.
Treatment objectives, therapeutic approach and the assessment of comorbidities, are shown in (Figure 1).

Treatment Objectives (ABCDEH):

A. Glycemic control (A1c)

General recommendation, which should always be individualized, is a target of HbA1c 7.5-8.5% (58-69 mmol/l) in advanced frailty, and HbA1c 7-8% (53-64 mmol/l) in mild to moderate frailty. In frailty subjects, HbA1c <7% (53 mmol/l) should be avoided, especially if drugs with risk of hypoglycemia are used [2]. Many frail subjects have medical conditions that can interfere with HbA1c determination (chronic kidney disease, anemia, transfusions), and capillary blood glucose measurement may be necessary for assessing glycemic control [2].

B. Blood pressure (BP)

The objective of elderly subjects with diabetes, including those with dementia, is <140/90 mmHg, avoiding values <120/70 mmHg. A goal of <150/90 mmHg may be more suitable for the frail and dependent elderly. Whenever possible, measure BP standing and sitting, to detect orthostatic hypotension that increase the risk of falls. Withdrawal of treatments should be evaluated as frailty progresses [2,3].

C. Hypercholesterolemia

Statin treatment is recommended in the same situations as in non-elderly subjects: secondary prevention and primary prevention with high cardiovascular risk. Treatment of hypercholesterolemia in elderly patients has some differential characteristics. Lifestyle changes may not be possible. Furthermore, statin myopathy is more frequent (up to 10%) due to sarcopenia, so it is advisable to use low or moderate doses of statins. Treatment of vitamin D deficiency can improve statin-associated myalgia [3]. In situations of advanced frailty and dependency, suspension of statins may be considered.

D. Assessment of chronic complications

It must be individualized, with particular attention to those with higher influence on functional state (retinopathy, diabetic macular edema and diabetic foot). Heart failure, chronic kidney disease, and vitamin B12 deficiency should not be forgotten [2,4].

E. Geriatric Evaluation

Consider the assessment of geriatric syndromes: polypharmacy (use of three or five drugs simultaneously or the need to indicate one drug to supply the side effects of another), cognitive impairment, depression (Yesavage scale annually), urinary incontinence, falls, chronic pain (visual analogue pain scale), and frailty [2,3].

F. Hypoglycemia

In older people prevention of hypoglycemia is especially important because of the repercussions on the risk of falls, fractures, and emergency department visits and hospitalization. Elderly patients have impaired recognition of hypoglycemia and difficulties in acquiring basic skills for self-care and for resolution of hypoglycemia, which determines a greater severity of the episodes. Also, there is a bi-directional relationship between hypoglycemia and cognitive decline [5].

Comprehensive Pharmacological Treatment in the Elderly with T2DM

In general terms, disease modifying therapies should be used in combination with metformin, that is, with benefit in morbidity - associated mortality, low risk of hypoglycemia, and benefits in terms of control of BP and excess of weight (if appropriate) [6].
The patient and their caregivers should be aware of the “sick days” rule for metformin and sodium-glucose transport protein 2 inhibitors (SGLT2i), to avoid the potential risk of impaired renal function, lactic acidosis, and euglycemic ketoacidosis. Also, simplification of complex regimens is recommended, especially in patients with insulin therapy, to reduce the risk of hypoglycemia and polypharmacy, always based on individualized HbA1c targets.
The use of SGLT2i in frail elderly patients with a diagnosis of heart failure (HF) with reduced ejection fraction (FEr), is a reasonable therapeutic option, given its potential benefits. Diuretic and blood pressure treatment must be revised to avoid volume depletion (hypotension, orthostatic hypotension, dizziness, syncope, and dehydration), and impaired kidney function.
DPP4 inhibitors (DPP4i) may be reserved for elderly people with renal function contraindicating other therapies, or those patients with normal weight, in whom the additional weight loss may be a problem; in this case, sitagliptin [7]. and linagliptin [8] must be prioritized. Sulfonylureas and glinides (hypoglycemia risk), and pioglitazone (risk of heart failure and fractures), must be avoided.
In frail elderly people with obesity, the use of weekly glucagonlike peptide-1 receptor agonists (GLP1ra) may be a good option given the low risk of hypoglycemia, the weight loss benefit, the potential benefits in comorbidities and the weekly administration [9]. Its use must be accompanied by adapted nutritional therapy, and appropriate physical activity recommendations (aerobic and resistance training) to avoid the loss of muscle mass, including strength, flexibility and balance exercises [10]. Also, the appearance of gastrointestinal effects should be monitored.

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Lupine Publishers| Identification of The Downregulation of TPD52-Like3 Gene and NKX2-1 Gene in Type 2 Diabetes Mellitus Via RNA Sequencing

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

A recent study using next-generation RNA sequencing was reported on genome-wide changes in gene expressing in the skin between patients with type 2 diabetes mellitus, compared to non-diabetic patients. Ex-post review, based in part on both the existence of lipid droplets, peridroplet mitochondria and cytoplasmic mitochondria, selected in the gene metabolism category the most downregulated gene TPD52L3, and in the gene regulation category the most downregulated gene NKX2-1. There is strong evidence that these two genes are involved in the disease process of type 2 diabetes mellitus.

Keywords: Gene Expression; Lipid Droplets; Mitochondria; RNA Sequencing; Type 2 Diabetes Mellitus

Opinion

In an earlier study, it was proposed that the final consequence of hereditary anomaly results in the development of type 2 diabetes, which already emerges in the prediabetic phase. It was thought to occur due to an increased flux, as compared to the healthy controls where protons (H+-ions) from the mitochondrial intermembranespace re-enter the matrix via uncoupling protein-1 (UCP1). This causes hyperthermia in and around the mitochondria [1].
But the key question that remains to be answered here is for the connection between the increased flow of protons and type 2 diabetes mellitus. In the past decade, a study reported on the visual documentation of the possible interaction of lipid droplets with mitochondria. This interaction was found to be quite intimate with the involvement of membrane attached receptor proteins such as SNAP23 [2]. Also, the cellular population of mitochondria in brown adipocytes tissue could be divided into two subpopulations; i.e. mitochondrial population having physical evidence of adherence to a lipid droplet or peridroplet mitochondria, and a non-lipid droplet-bound cytoplasmic mitochondrial population without any adherence to lipid droplets [3,4].
Although both the peridroplet mitochondria and cytoplasmic mitochondria are similar in their cell membrane composition they differ in other fundamental respects [3,4]. A comparison of the purified peridroplet mitochondria to cytoplasmic mitochondria suggests that peridroplet mitochondria are more elongated, whereas cytoplasmic mitochondria tend to be smaller. Also, peridroplet mitochondria have enhanced oxidative phosphorylation capacity, TCA cycle activity, ATP synthesis, as well as increased ATP-dependent triglyceride synthesis compared to cytoplasmic mitochondria. The measured fatty acid-driven respiration and UCP1 content in the isolated mitochondria suggests that for thermogenic fat oxidation peridroplet mitochondria are not specialized compared to cytoplasmic mitochondria [3]. This signifies that, under healthy conditions, in the peridroplet mitochondria the protons derived from free fatty acids (FFAs) and generated by the electron-transport chain during the oxidation process of FA are used for the production of ATP without any escape of protons via UCP1 to produce heat. On the other hand, the protons generated by the oxidation of cytoplasmic mitochondrial FA are mainly used for the production of heat. So, peridroplet mitochondria have an increased coupled respiration, while cytoplasmic mitochondria have an increased uncoupling activity. The existence of peridroplet mitochondria demonstrates that the essential processes of fat metabolism can be selectively confined to exclusive and segregated subsets of mitochondria. The fatty acids intended for storage undergo synthesis of triacylglycols followed by their storage in the lipid droplets.

Most eukaryotic cells can store lipids in the form of droplets [5]. Lipid droplets are cytosolic storage organelles at the center of the lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, mostly triacylglycerol and sterol esters and are enclosed by a phospholipid monolayer membrane. This single layer is derived from the endoplasmic reticulum, whereby triacylglycerols are synthesized between the two leaflets of the endoplasmic reticulum membrane. Associated with the monolayer is a specific set of proteins, which decorates the surface of the lipid droplet but is absent from the hydrophobic core [6]. These proteins associate with the membrane through hydrophobic hairpins, amphipathic helices and fatty acid modifications, and are also thought to control lipid droplet positioning inside the cell and association with other organelles.
In 2016, researchers demonstrated that the exogenous expression of human tumor protein D52 (TPD52) in the cultured 3T3 cells result in a significant increase in the numbers of lipid droplets [7]. Starting with the bulging of a triglyceride lens within the endoplasmic reticulum bilayer, lipid droplet biogenesis factors including TPD52 are recruited to the lens structure and facilitate the growth of the nascent lipid droplet [8,9]. Moreover TPD52- expressing 3T3 cells form more lipid droplets following oleic acid supplementation, which contributes to the lens formation [10]. As a previous study has shown, an increase in carbon-carbon double bonds in the acyl chains of phospholipids promotes the flexibility of cellular membranes [11]. So, TPD52 expression increases lipid storage, co-distributes with lipid droplets and is recruited to lipid droplets to stabilize lipid droplets [12]. Moreover, it is interesting to note that TPD52 knockdown decreased both lipid droplet sizes and numbers [12].
Tumor protein D52 is the founding member of the TPD52-like protein family representing four paralogous mammalian genes, i.e. TPD52, TPD52L1, TPD52L2, and TPD52L3 [7,13,14]. The group of Cao demonstrated that human TPD52L3 interacted with itself and with TPD52, TPD52L1, and TPD52L2 [14]. The four human linear proteins of this family are TPD52, TPD52L1, TPD52L2, and TPD52L3, which consist of 184, 204, 206, and 140 amino acid residues, respectively. Their first exon-coded protein located at the N-terminal side is unique to each isoform, and all the members contain a highly conserved coiled-coil motif located towards the N-terminus which is required for homo- and heteromeric interaction with other TPD52-like proteins [14,15] and share a sequence identity of ~50% [7]. Byrne et al. proposed that TPD52 may exert and/or regulate its activities through interaction with itself and its related proteins [15]. The coiled-coils were predicted by use of pairwise residue correlations and were not based on their crystal structures [16].

Figure 1: Alignment of the human TPD52 (upper row) and the human TPD52L3 (lower row) protein sequences. Amino acid residues are indicated by single letters. Vertical lines indicate identical residues and colons/dots indicate highly/weakly conserved residues.

The analysis was focused on the sequences of TPD52 and TPD52L3. The two sequences revealed an obvious similarity, they share 63 identical positions and 42 similar positions resulting in an overall homology of 57.1% (Figure 1). The coiled-coil motif near the N-terminus was found in all TPD52-like proteins. Uniprot predicts a coiled domain for residues 28-57 of TPD52L3, and also for residues 21-70 of TPD52 [17]. The common part of these two coiled-coil protein sequences has an overall homology of 67.9% (Figure 1). The information regarding the amino acid sequences of human TPD52 (P55327-2) and TPD52L3 (Q96J77-1) was retrieved from the UniProtKB/Swiss-Prot databank. Interestingly, although the coiled-coil motif is very important for the interactions mediated by TPD52-like proteins [15,18], the TPD52L3 shortened coiled-coil motif successfully interacted with TPD52-like proteins.

A recent study using next-generation RNA sequencing was reported on genome-wide changes in gene expression in the skin between patients with type 2 diabetes mellitus, compared to nondiabetic patients [19]. The most downregulated gene of patients with type 2 diabetes in the gene metabolism category is TPD52L3 with a “log2 fold change” value of -28, compared to skin samples from non-diabetic patients. So far, this gene has not been linked to type 2 diabetes or wound healing.

The fact that the exogenous expression of human TPD52 increases the number of lipid droplets [7], TPD52 knockdown decreases the number of lipid droplets [12], and the activity of TPD52 depends on the interaction with TPD52L3 [14], support the idea that the major function of TPD52L3 is the lipid storage at the center of the lipid and energy homeostasis [8,9]. In other words, in brown adipocytes tissue, it seems likely that the significant downregulation of TPD52L3 causes a reduction in the number of lipid droplets in the skin samples of type 2 diabetes mellitus patients.

This indicates that an essential reduction in the lipid droplets suggests a substantial decrease in the peridroplet mitochondria for patients with type 2 diabetes and consequently an increase in the saturated plasma FFAs. As the unsaturation index (UI; number of carbon-carbon double bonds per 100 fatty acyl-chains) of FFAs from human white fat cells is substantially lower compared to the UI of serum FFAs in the healthy controls (85.5 and 191.9, respectively), these events force a shift from unsaturated to saturated acyl chains in the phospholipids of both the erythrocyte and vascular membranes [20]. This reduction in UI translates into an increase in the attractive forces between the mutual membrane phospholipid acyl chains, which redistributes the lateral pressure profile of the cell membrane [21]. This redistribution reduces the pore diameter of the transmembrane glucose transport channels of all Class I glucose transporter proteins, leading to a marked reduction in the transmembrane glucose transport [22].

On the other hand, the increased uncoupling activity of the cytoplasmic mitochondria [4] takes up the remaining fatty acid oxidation, including the formation of protons. The rationale is that the overall balance between the number of protons which re-enter the matrix through ATP synthase on the one hand, and the number of protons which re-enter the matrix through UCP1 on the other hand, might shift to the latter side, which in turn promotes an increase in the production of heat. To keep a narrow range of mitochondrial temperature compatible with life, the slow-down principle enters into force, which also results in an essential reduction in UI [1]. This chain of events is a blueprint of the development of type 2 diabetes mellitus.

A second result of the earlier mentioned genome-wide analysis study is the most downregulated gene in the gene regulation category, NKX2-1, of type 2 diabetes patients with a “log2 fold change” value of -28 compared to skin samples from non-diabetic patients [19]. Notably, a study also reported a novel heterozygous mutation in exon 3 of the NKX2-1 gene, which is related to a reduction in the muscle mitochondrial respiratory chain complex activity, a characteristic of type 2 diabetes [23]. It is to be noted that the reduced mitochondrial activity is one of the characteristics of type 2 diabetes [24]. This may be in advance of the patients with type 2 diabetes mellitus as the reduced mitochondrial activity implies a reduction in heat production.

Finally, it is worth considering about the potential benefit of the use of (modified) synthetic TPD52L3 for combating the adverse effects of type 2 diabetes mellitus.

Briefly, the idea is that two genes are pertinently involved in the disease process of type 2 diabetes mellitus: one concerns the downregulation of human TPD52L3 gene expression, which yields a significant reduction in the lipid droplets, whereas the second one relates to the downregulation of human NKX2-1 gene expression which reduces the mitochondrial respiratory chain activity (Figure 2).

Figure 2: Although the results of genome-wide screen for type 2 diabetes susceptibility genes are still under debate, a refined working hypothesis proposes that the primary effect of the downregulation of the human genes TPD52L3 and NKX2-1 generates an increased flux of mitochondrial intermembrane-space protons through UCP1 into the matrix, which causes an increase of extra heat. This process initiates the slow-down principle. UCP: Uncoupling protein; FFA: Free fatty acid; GLUT: Glucose transporter.  

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Lupine Publishers| Properties of Mitochondrial-Derived Peptides (Mdps), Type 2 Diabetes, and Relationship with Oxidative Stress

 Lupine Publishers| Journal of Diabetes and Obesity

Abstract

Objective: In addition to its role in energy production and metabolism, mitochondria play a major role in apoptosis, oxidative stress, and calcium homeostasis. This review highlights the intricate role of mitochondria derived peptides (MPs), oxidative stress, and age-related disease such as diabetes.

Key Findings: The mitochondria produce MDPs: specific peptides that mediate transcriptional stress response by its translocation into the nucleus and interaction with DNA. MDPs are regulators of metabolism with cytoprotective effects through anti-oxidative stress, anti-inflammatory responses and anti-apoptosis. This class of peptides comprises: humanin (HN), MOTS-c, Small HN-like peptides. HN inhibits mitochondrial complex 1 activity and limits oxidative stress level in the cell. HN has been shown to prevent apoptosis by decreasing the reactive oxygen species production. Mitochondrial dysfunction and oxidative stress are implicated in the pathogenesis of diabetes. Data suggested that MDPs had a role in improving type 2 diabetes (T2D).

Summary: The goal of this review is to discuss the newly emerging functions of MDPs and their biological role in ageing and age-related diseases such as T2D.

Keywords:Mitochondrial-Derived-Peptides; Humanin; Oxidative Stress; Diabetes

Introduction

Mitochondria play a critical role in maintaining cellular function by ATP production. In addition to its role in energy production and metabolism, mitochondria play a major role in apoptosis, oxidative stress, and calcium homeostasis. A mitochondrial stress signal, or a ‘mitokine’, could confer protection and promote survival, while priming the cell’s readiness for subsequent insults with increasing severity. The term ‘mitohormesis’ for such a phenomenon has been created [1]. The mitochondrial unfolded protein response (UPRmt) is a central part of the “mitohormetic” response. The UPRmt may be an alternative way in relationship with mitochondria signal in the cell. The mitochondria produce some specific peptides that mediate transcriptional stress response by the translocation into the nucleus and interaction with DNA. Mitochondrial derived peptides (MDPs) are regulators of metabolism and various studies have shown that MDPs exerted cytoprotective effects through anti-oxidative stress, anti-inflammatory responses and anti-apoptosis [2,3]. The goal of this review is to discuss the newly emerging functions of MDPs and their biological role in ageing and metabolic diseases such as T2D.

Mitochondrial Metabolism Modulation

Functions in the Mitonuclear Communication Pathways

Mitochondria booked a portion of the original bacterial genomes that co-evolved with nuclear genome. However, mitochondria import over a thousand proteins encoded in the nuclear genome to maintain their diverse functions, reflecting their adjacent relationship [4].

The mitochondrial genome inherits bacterial-like traits: the DNA molecules (mtDNA) are circular, double stranded, small (16,569 nucleotides in humans) and compact. mtDNA contains 37 genes, including 22 tRNAs, 2 rRNAs (12S and 16S rRNA) and 13 mRNAs encoding the proteins of the electron transport chain [5]. The mtDNA has no introns but a few non-coding nucleotides between adjacent genes and small open reading frames that encode functional MDPs. This class of peptides comprises humanin (HN) and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) and expands the expression of mitochondrial proteome [6]. It has been established that mitochondria can export peptides and also import cytosolic peptides. It is the class of “cell-penetrating peptides” designed also as “mitochondrial cell-penetrating peptides” [7]. Many age-induced processes and degenerative diseases are related to mitochondrial dysfunction, further highlighting the critical importance of this organelle [8]. Complex human diseases, including diabetes, obesity, fatty liver disease and aging-related degenerative diseases are associated with alterations in mitochondrial oxidative phosphorylation (OXPHOS) function.

Overview on Concepts of Retrograde Signaling and Unfolded Protein

Numerous implications of these anterograde and retrograde signaling pathways between the mitochondria and the nucleus are appropriate for therapeutic exploitation with bioactive molecules.

Concept of Retrograde Signaling

The hallmark of mitochondrial retrograde signaling is the modification of the expression of nuclear genes induced by a signal from mitochondria [9]. Retrograde signaling must be triggered by a mitochondrial signal that in turn is relayed via molecules that finally reach the nucleus. In mammalian cells, altered nuclear expression in response to mitochondrial dysfunction is reported; a number of signaling pathways being implicated in this retrograde communication [10]. Mitochondrial retrograde signaling is a signaling pathway connecting mitochondria and the nucleus. Signal transducers in the yeast retrograde response are Rtg1p, Rtg2p, and Rtg3p proteins [11]. The outcomes of mitochondrial retrograde signaling go far beyond the maintenance or biogenesis of the organelle, affecting the homeostasis of the whole organism through body weight or immunity.

Concept of Unfolded Protein

Mitochondrial protein homeostasis is maintained through proper folding and assembly of newly translated polypeptides. Several factors challenge the mitochondrial protein-folding environment including reactive oxygen species (ROS) that are generated within mitochondria, as well as environmental situations such as exposure to toxic compounds. To promote efficient mitochondrial protein folding mitochondria possess molecular chaperones located in both the intermembrane space and matrix [12].

UPRmt is a mitochondria-to-nuclear communication mechanism that promotes adaptive regulation of nuclear genes related to mitochondrial response, and metabolism, implicated in the cellular homeostasis [13].

Mitochondrial-Derived Peptides: Classification

MDPs are a series of peptides encoded by mitochondrial DNA. This class of peptides comprises HN, MOTS-c, Small HN-like peptides (SHLPs) and expands the expression of mitochondrial proteome [6].

Humanin

The first MDP discovered back in 2001 was HN; the term based on the potential of this peptide for restoring the “humanity” of Alzheimer’s disease (AD) patients. HN promotes cell survival in response to a variety of insults.
It is a small, secreted, 24 or 21 amino acid peptide, depending on cytoplasmic or mitochondrial translation, respectively. If HN is translated within the mitochondria, the peptide will be 21 amino acids; and if it is translated in the cytoplasm, then the result is a 24 amino acid peptide [14]. HN is encoded by an HN open reading frame (ORF) within the gene for the 16S ribosomal subunit within the mitochondrial genome [15]. HN was discovered during a search for survival factors in unaffected areas of an AD patient’s brain. The initial studies were first performed in cell culture and then followed by in vivo studies using both pharmacological mimetics of AD as well as mutant gene: amyloid-β precursor protein. The most recent studies used transgenic models of AD. As HN is a relatively short peptide, exhaustive mutational analysis of the importance of each amino acid has been possible. Interestingly, single amino acid substitutions of HN can lead to significant alterations in its potency and biologic functions. S14G-HN in which the serine at position 14 is replaced by glycine, is a highly potent analogue of HN.
Finally, HN may be the first small peptide of its kind representing a putative set of MDPs, a novel concept that modifies the established concept about retrograde mitochondrial signaling as well as mitochondrial gene expression. HN is a neuroprotective peptide and a cytoprotective factor against oxidative stress [16].

Mitochondrial Open Reading Frame of the 12S rRNA-c ( MOTS-c)

In addition to HN, an in-silico search of the mitochondrial genome revealed additional potential MDPs. MOTS-c is expressed in various tissues and in circulation (plasma) in rodents and humans, suggesting both a cell-autonomous and hormonal role. Its primary target organ appears to be skeletal muscle and fat. The mitochondrially derived peptide MOTS-c was recently discovered. It is a 16 amino acid peptide located in the 12S rRNA gene. The first 11 amino acid residues of MOTS-c are highly conserved in 14 mammalian species [17]. MOTS-c has been identified as a gene expression regulator in the nucleus, leading to retrograde signaling via its interaction with transcription factors. MOTS-c polymorphism has been found to be associated with human longevity [18]. MOTS-c can prevent insulin resistance, dietmediated obesity, and ameliorate diabetes. MOTS-c oxidizes fatty acids and inhibits oxidative respiration [19]. MOTS-c increased the levels of carnitine metabolism, which transport activated fatty acids into the mitochondria for β-oxidation, increased the level of a β-oxidation intermediate. MOTS-c inhibited the folate cycle at the level of 5Me-THF, resulting in an accumulation of 5-aminoimidazole- 4-carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator. MOTS-c also increased cellular NAD+ levels, which are nucleotide precursors [17,20]. MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors [21].

Small HN-like Peptides

Recently, six additional peptides encoded within the mitochondrial 16S rRNA region of mtDNA have been discovered and designed as SHLP1-6. SHLP2 and SHLP3 share similar biological effects with HN. The circulating levels of MOTS-c and SHLP2 decline with age. Various studies suggest that SHLP2 and SHLP3 may participate in the pathogenesis of age-related neurodegenerative diseases. The anti-oxidative stress function of SHLP2, and its neuroprotective effect indicate that SHLP2 has a role in the regulation of aging and age-related diseases [5].

Ageing and Plasma MDPs Levels

Ageing and longevity are or are not characterized by high levels of MDPs? It is speculated that MDPs production turns from protective to detrimental adaptive response; in these conditions, the levels increasing during aging. In some studies, HN levels significantly decline with age in humans. Plasma HN level was significantly lower in the older group (1.3 ± 0.2 ng/mL) than that of the younger group (1.7 ± 0.1 ng/mL) [22]. In other studies, it is reported that HN levels significantly decline with age in humans and animals. HN levels in plasma were measured in young and old mice and across age in humans. HN levels decreased with age in both mice and human (Human plasma levels: 45-65 years: 1400 pg/mL; 80-110 years: 1000 pg/mL) [5].

New results are in contrast with these data. HN plasma levels are evaluated in 693 subjects aged from 21 to 113 years. HN levels increased in old age (>500 pg/mL), with the highest levels found in centenarians (> 1000 pg/mL). The plasmatic levels of HN are significantly positively correlated with age. No gender differences were observed for HN. HN plasma level is associated negatively with body mass index in elderly patients [23]. Concerning the other MDPs, it is reported that MOTS-c and SHLP2 circulating levels decline with age. The circulating SHLP2 levels significantly decreased with age in both male and female C57BL/6 mice (young, 3 months old: 3000 pg/mL; aged, 18 months old: 2500 pg/mL). Male mice had higher SHLP2 levels than female mice in both the young and old groups [5]. The results of these studies should be interpreted considering the following limitations. First, the relatively small sample size in each group represents a potential limitation. Second, mitochondrial diseases are an expanding group of disorders with many metabolic deficiencies. In the ideal case, the used patient cohort should display a homogeneous phenotype, disease stage, and organ specificity. Moreover, the discovery of ageing-related biomarkers is supported by the development of advanced proteomics technology. Changes in the circulating concentrations of human proteins can serve as predictive measures of health and disease [24].

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Happy Thanksgiving!

 

Today is the time to be thankful, remember good times, and embrace those who enrich our lives. I’m thankful for a lot of things. Happy Thanksgiving to all! from our Archives of Diabetes and Obesity (ADO)

Lupine Publishers | Variability in Plasma FGF21 Levels in Rats Fed A Standard 15% Protein Diet is not Sensitive Enough to Reflect Differences in Protein Requirements

 Lupine Publishers | Journal of Diabetes and Obesity

Introduction

Fibroblast growth factor 21 (FGF21) is a hepatokine member of a subfamily of “fibroblast growth factors” that responds to multiple metabolic stresses as protein deficiency [1-4]. FGF21 is produced in various tissues but the FGF21 circulating form is primarily of hepatic origin [1,2]. FGF21 affects numerous metabolic and behavioural parameters, and in particular, increases appetite for protein in subjects fed a protein-deprived diets [5,6]. In a recent still unpublished study, we observed that plasma FGF21 levels were higher in adult male Wistar rats fed a standard diet, formulated according the AIN93 recommendations for rats’ feed, containing 15% protein by energy [7] than in rats fed a 30% protein diet. In addition, inter-individual variability of plasma FGF21 levels was larger in rats fed the standard 15% protein diet than in rats fed the 30% protein diet. We therefore considered the hypothesis that higher levels and inter-individual variability in plasma FGF21 levels in rats fed a standard 15% protein diet would reflect the variability in protein requirements between individuals and thus, that measurement of plasma FGF21 levels can be used as a simple, rapid, and minimally-invasive test to estimate the adequacy of protein intake.
Dietary self-selection is a method that has been largely used in farm animals and laboratory rodents to study the requirements for macronutrients (carbohydrates, lipids and proteins), vitamins and minerals [8,9]. Many studies using this method, in our lab and others, showed that rats self-selecting between a protein diet and a protein-free diet often ingest up to 30-50% of total energy intake as protein [10-15], so much higher than the level considered as sufficient for an optimal growth in adult rats (10-15% by energy), which comforted our hypothesis that 15% dietary protein was possibly not the optimal dietary content.
The objective of this study was to verify that variability in plasma FGF21 levels in rats fed a standard 15% protein diet was indicative of differences in protein requirements. To this end, we have analyzed the relationship between FGF21 levels, and the level of protein subsequently selected during self-selection between a protein diet and a protein-free diet.

Experimental Procedure

24 adult male rats (215-240g) of the Wistar RccHan strain (ENVIGO) were used and individually housed (22°C ± 1°C, 12/12 L/D, cycle lights on at 08:00). After 1 week of adaptation to the laboratory conditions, the rats were fed for 12 days (Basal period) a standard diet formulated according to the AIN93 requirements [7] that contained 15% protein (15P); then, for 28 days (Choice period), 6 rats (Control group) continued to be fed with the standard diet and 18 (Self-selecting group) were given a choice between a pure protein diet (100P) and a protein-free diet containing a mix of fat (soy oil) and carbohydrate (corn starch and sucrose) in which carbohydrate amounted 60% by energy. The diets were provided, as necessary.

The food pellets were prepared twice a week by mixing the macronutrients, vitamins, and mineral mix with the amount of water required to make a thick dough. Food intake (g/day) was measured twice a week and converted in kJ/day based on the energy content of the diets (Table 1).

Table 1: Composition and energy content of the 3 used diets.

100P: diet containing only proteins; 60C: protein-free diet containing only lipids and carbohydrates and in which carbohydrates amounted 60% by energy; 15P: standard diet containing 15% of protein by energy.

Blood samples (0.5 mL) were collected from the tail vein in EDTA tubes: once during the basal period and once during the choice period. Blood collection was made in the morning (10:00- 12:00) in rats that were not previously fasted. Blood samples were centrifuged (5000g, 15min, 4°C) and the plasma stored at -20°C. Plasma FGF21 levels (pg/ml) were measured by ELISA tests using commercial kits from Bio Vendor (Mouse/Rat FGF-21 ELISA RD291108200R).

Statistical Analysis

Statistical tests were performed using RStudio software, 2015. Changes in protein intake and plasma FGF21 level were compared using mixed two-factor ANOVA tests (parameter ~ group*period), which were followed by the main effects analysis by Bonferroni adjusted pairwise comparisons. Values are presented as means ± standard error of the mean (SEM). Linear regression analysis was used to study the link between plasma FGF21 levels during the basal period and protein intake during the choice period and was performed using Excel software. Significance of correlations was assessed using the Pearson correlation coefficient. A threshold of P≤0.05 was chosen as significant.

Results and Discussion

Protein intake was similar between the control and selfselecting group during the basal period but increased by 80% in the self-selecting group during the choice period (+37.8 kJ/d, p<0.0001) (Figure 1). This response significantly increased the contribution of protein to total energy intake from 15.0% to 23.5% (p<0.001). Mean plasma FGF21 levels averaged ~1,100 pg/mL in both groups during the basal period and decreased to 131 pg/mL in self-selecting group during the choice period (P<0.001) (Figure 2). Finally, contrary to our hypothesis, not only did we not observe a positive correlation between plasma FGF21 levels during the basal period and protein intake during the choice period, but instead we observed a weak and inverse correlation (Figure 3).

Figure 1: Protein intake (kJ/d) according to diet group and period.
(*:0.05; **:0.01; ***:0.001; ****:0.0001) Values are represented as means ±SEM, only the p-value of the interaction of ANOVA tests are indicated.

Figure 2: FGF21 level in plasma (pg/ml) according to diet group and period.
(*:0.05; **:0.01; ***:0.001; ****:0.0001) Values are represented as means ±SEM, only the p-value of the interaction of ANOVA tests are indicated.

Figure 3: Protein intake (kJ/d) during the choice period as a function of plasma FGF21 levels during the basal period in the self-selecting group.

Conclusion

In conclusion, inter-individual variability in plasma FGF21 levels in rats fed a standard 15% protein diet did not appear to be a parameter sensitive enough to reflect inter-individual differences in protein requirements. Therefore, plasma FGF21 level cannot be used as a test to determine inter-individual variability in protein requirements in individuals. Nevertheless we observed that plasma FGF21 levels in P15 fed rats were ~7 fold higher than in selfselecting rats ingesting 23.5% protein, which points on the fact that changes in plasma FGF21 levels are very sensitive to dietary protein intake, even when protein intake is well above essential protein requirements (~8-10 % in adult male rats).

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Lupine Publishers| Excess Weight, Obesity, Diabetes and Coronavirus Disease

 Lupine Publishers| Journal of Diabetes and Obesity

Introduction

SARS-CoV-2 virus has created an unprecedented public health and global economic crisis. Despite the fact, that this virus was discovered half a century ago, earlier version of this RNA virus (SARS-CoV) was less virulent than the 2019 version of the virus (CoV-2), which turned out to be the most potent killer virus. According to the Johns Hopkins University (JHU) Coronavirus Resources Center (coronavirus.jhu.edu), globally there are 28 million Covid-19 infected individuals and 900,000 deaths. In the USA, we have over 6.4 million infected individuals and 192,000 confirmed covid-related deaths. The response from the public health perspective is important, to prevent further escalation of the SARS-CoV-2 epidemic. Having said that, it is important to note, that each country has responded differently. Vietnam, and Taiwan have done well in keeping the Covid-19 infection rate and death to a minimum. Public health experts worldwide, should study the response of Taiwan, in preventing the spread of this highly contagious disease, without massive tests and lockdowns. Currently the oldest (USA) and the youngest (India) democracies, are competing for the number one position, interns of highest number of infected individuals. India has over 4.5 million infected individuals and 76,000 deaths. According to the JHU report dated September 10, 2020, Mexico has the highest observed case-fatality ratio (CFR) of 10.7%, compared to the USA (3.0%), and India (1.7%). In earlier articles we have discussed the role of comorbidities such as hypertension, excess weight, obesity, diabetes (Type-2) and vascular diseases, on the severity of Covid-19 infection [1-6]. However, if one looks at this situation from a historic perspective, in the last four decades cardiometabolic disease such as hypertension, excess weight, obesity, diabetes (Type-2), and vascular diseases, have increased in prevalence and incidence to epidemic proportions worldwide [7-16]. Added to this global burden of metabolic diseases, a killer virus has taken advantage of the existence of these metabolic risks, which are known to promote, oxidative stress, inflammation, vascular and immune dysfunction.

China Medical Treatment Expert Group for Covid-19, reported in January of 2019, that on admission, 20-51% of patients reported as having at least one comorbidity, with diabetes (10-20%), hypertension (10-15%), and other cardiovascular diseases (7-40%) being the most common [17]. In this guest editorial, we will briefly discuss the role of metabolic diseases such as hypertension, obesity, and diabetes in the progression and severity of the coronavirus disease. According to news reports, the first group of people to get hit by the virus in Italy were the elderly. They also noted that Italy has the second oldest population in the world, after Japan. Irony of this comparison is, that Japan did not suffer such devastating effect from the SARS-CoV-2 as Italy and Spain. In China, of the 1590 patients hospitalized in the early days of Covid-19 pandemic, the mean age was 48.9 years. In the USA, rates were highest among persons aged 65 years (12.2%), 65-74 (17.2%), and population older than 85 (54.4%). Public health experts believed that younger population was less susceptible for the SARS-CoV-2 infection. As is with any prediction about the Covid-19, the story keeps changing as the timeline changes. Currently there are over 500,000 infected young students in the USA. Earlier reports from China, indicated hypertension and diabetes (Type-2) as the two major comorbidities. However, in the recent months, dozens of studies have reported that many of the sickest patients have been people with obesity [18]. Furthermore, studies have demonstrated that even people who are merely overweight, also are at higher risk for Covid-19. The study by Ogden et al reported, the prevalence of childhood and adolescent obesity, and noticed grater increases (2-fold) in non-Hispanic Black and Mexican American adolescents [19]. This is particularly concerning, because adolescents with severe obesity are at high risk for the development of serious comorbidities including hypertension, diabetes as well as Covid-19. In a recent editorial in JAMA, Rodgers and Gibbons discuss the role of obesity and hypertension as comorbidities of Covid-19. SARS-CoV-2 pandemic has brought out the susceptibility of minority communities of color, and has exposed the complex interplay of contributing factors, that are rooted in the social determinants of health, and racial inequities. A 6-fold increase in the rate of death for African Americans, living in the USA due to a ubiquitous virus should be deemed unconscionable, as reported in the recent issue of JAMA (April15, 2020). What is currently known about these differences in disease risk and fatality rates? In Chicago, more than 50% of COVID-19 cases and nearly 70% of COVID-19 deaths involve African American individuals, although they make up only 30% of the population. This trend can me tracked down in various US Cities. Poor living conditions, health care disparity, unhealthy nutrition, and high incidence of metabolic diseases, seem to contribute to the excess CFR in this ethnic group, as well as in other minority communities [20, 21].

Considering the contribution of comorbidities to the progression and severity of the coronavirus disease, one would expect that China and India, with the largest populations of diabetic subjects, should have the highest CFR (Deaths per 100,000 population) for Covid-19. On the other hand, Mexico (10.7%), Iran (5.8%), and Spain (5.4%) have lot more mortality than the USA (3.0%) and India (1.7%). Since the two major populations with highest number of diabetics have not shown comparatively high case fatality rate, it is worthwhile discussing the other two comorbidities (hypertension and obesity) as the chief contributors for the Covid-19 progression and severity. Trends in the prevalence of hypertension in the USA, according to the NHANCE survey of age standardized prevalence, decreased from 48.4% in 1999-2000 to 45.4% in 2015-2016. However, absolute burden of hypertension consistently increased, from 87.0 million in 1999-2000 to 108 million in 2015-2016 [22]. Hypertension appears to be more common in Mexico, than among Mexican Immigrants in the United States. As far as the obesity goes, the number of obese children and adolescents aged five to 19 years, has risen tenfold in the past four decades and if current trends continue, there will be more obese children and adolescents than those moderately or severely [23]. Among adolescents, obesity prevalence in the USA was 16.8% in 2007 and 18.5% in 2016. Age standardized obesity in adults increased from 33.7% in 2007 to 39.6% in 2015. Whereas, 62% of the participants in Mexico reported, at least, being overweight [24]. When considering obesity data based on the BMI, we should keep in mind that South Asians have a different body fat distribution, compared to the European and Western population. South Asians in general have central abdominal obesity.

Data from 6916 patient records that researchers from Kaiser Permanente reported, compared to normal body mass index (BMI) of 18-24 Kg/m2, the risk of death more than doubled for patients with a BMI of 40-44 Kg/m2 and nearly doubled again, for those with a BMI of 45kg/m2 or more [25] In an accompanying editorial, David A Kass, a Cardiologist at the Johns Hopkins University, wrote, “that these findings taken with prior research -should put to rest the contention that obesity is common in severe COVID-19, -because it is common in the population.” The pathophysiology of hypertension involves, complex interaction of multiple vascular effectors, including activation of the sympathetic nervous system, of the renin-angiotensin-aldosterone system, and of the inflammatory mediators. Oxidative stress and endothelial dysfunction are consistently observed in hypertensive subjects [26]. As we have discussed earlier, obesity has reached epidemic proportions worldwide. In the USA alone, the prevalence of obesity has increased 50% in the past three decades, with 70% of all adults being classified as either overweight or obese [27]. Beyond an impaired response to infections, people with obesity also suffer from chronic, low grade inflammation. Fat cells secrete inflammation triggering chemical messengers called cytokines, and more come from immune cells called macrophages, that clean up dead and dying fat cells. These in turn, impair vascular homeostasis and lead to endothelial dysfunction [28].

If we carefully analyze a series of clinical events, that develop post SARS-CoV-2 infection, we can begin to understand, why metabolic diseases serve as independent risk factors for the progression and severity of coronavirus disease. Initial route of entry is via nasal and oral mucosa, -the preferred receptor that facilitates the transmission seems to be the ubiquitous ACE2, which is found in multiple types of cells and tissue including vascular endothelium. Recent findings, that following the injury to the lung tissue, the virus gets entry into the endothelium, opens a whole new avenue for the progress of the disease and its severity. Endothelium is the largest organ of the body, covering a large surface area and reaching out to every tissue and organ. As such, the injury to the endothelium could introduce a cascade of events, leading to platelet activation, thrombin generation, and promotion of both thrombotic and thrombolytic events [3]. Furthermore, people with metabolic diseases such as, hypertension, excess weight, obesity, diabetes (Type-2), and vascular disease, already have a compromised endothelium and invasion of the SARS-CoV-2 virus leads to further injury to the vascular system, by the disruption of vascular integrity and endothelial cell death. These events lead to the exposure of the subendothelial basement membrane, and results in the activation of thrombotic and clotting cascade of events.

The question of why China and India with the largest populations of diabetics, have relatively low rates of Covid-related mortality, is quite puzzling. In China, -Covid-19 pandemic’s epicenter, Wuhan, and its province, Hubei, Chinese Center for Disease Control-network, formed 1300 epidemic investigation teams, in addition to the 40,000 doctors and nurses. They used very clever tracing tools with big data support. In the first week of January the novel coronavirus infection was detected, and on 23 January 2020, they locked down the city of 11 million people and soon the rest of the Hubei-a province of nearly 60 million. The WHO-China Joint Mission on Coronavirus Disease 2019 Task Force concluded, “In the face of unknown virus, China has rolled out perhaps the most ambitious, agile, and aggressive, disease containment effort in history.” The strategy that underpinned this containment effort was initially a national approach, that promoted universal temperature monitoring, masking, and hand washing [29]. As far as India is concerned, the general population thinks, that they have innate immunity, as they are exposed to a variety of Asian viruses. On the other hand, some scientists speculate that the SARS-CoV-2 in India is a milder version, compared to the European and US strains. According to a news report by Rajesh Nair in ‘The Hindu’ of September 11, 2020, “Diabetes seems to be the main cause of COVID-19 deaths in the Union Territories (UT) of India. A survey conducted by the Jawaharlal Institute of Postgraduate Medical Sciences and Research (JIPMER) showed 30% of the government servants in Puducherry (UT) were diabetic.

In the same report by Nair, Emergency Surgeon of New Medical Center, Dr T. Arjun Sundaram expresses his optimism by saying, “It is an obedient (SARS-CoV-2) virus, if treated early for even people with comorbidities. But people with comorbidities try to ignore early symptoms, as part of their existing medical conditions, - just as flu-like symptoms.” Comorbidities such as hypertension, excess weight, obesity, diabetes, and vascular diseases increase COVID-19 related hospitalization by 6-fold and deaths, by 12-fold. In a recent report from the USA, underlying conditions were reported in 71% of individuals admitted to hospital with COVID-19 and in 94% of the deaths [30]. In a study done at Westchester County, New York, among Covid-19 patients, who presented with a comorbid condition, more than 57% had high blood pressure, while 41.7% were obese and 33.8% had diabetes. This study also found 90% of coronavirus patients, who were put on ventilators died. A recent global estimate published in Lancet, estimated that one in five individuals worldwide are at risk for infection by SARS-CoV-2 virus. A recent report by Jain and associates from New Delhi, India, discusses differential mortality in COVID-19 patients from India and Western Countries [31]. The authors discuss the age of the population, genetics of the virus, mutation of the virus, immune variations of Indian subjects and the expression of the ACE2 receptor in the adipose tissue.

Authors claim that they have investigated and identified the possible reasons and hypotheses for this disparity in observed or reported Covid-19 related mortality. However, we feel strongly, that there may be other, as yet unknown causes, and only future history will reveal all the mysteries of coronavirus disease.

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