The aim of this study is to investigate possible enduring effects of a standard 2-month weight loss program on appetite regulation, bone homeostasis and muscle strength in younger and older adults, as well as the impact of differences in dietary composition during weight maintenance.

This sub-study consists of 2 parts:

PART 1. Durability of changes in appetite and appetite-regulating hormones after weight loss in overweight or obese pre-diabetic adults: impact of diet during weight maintenance.

A major reason for the failure of many lifestyle-based weight loss attempts is that the body responds to energy restriction and weight loss with a series of adaptive responses that prevent ongoing weight loss and promote weight regain (Sainsbury and Zhang 2010; Sainsbury and Zhang 2012). This 'famine reaction' includes increased appetite and alterations in circulating concentrations of gut-derived hormones that tend to stimulate appetite and promote fat accumulation. Recent evidence suggests that these alterations are sustained for as long as the lower body weight is maintained (Sumithran et al 2011). This finding - if validated in other populations - has enormous implications for clinical practice. For instance, if we know for certain that the weight loss-induced increase in appetite cannot be reversed without weight regain, or if we can identify those individuals for whom the increase in appetite does not subside, then multiple fruitless and heartbreaking efforts to lose excess weight via lifestyle interventions could be circumvented, and those individuals could immediately be channelled into more aggressive - albeit still imperfect - treatment options (i.e. long-term appetite-suppressing medications, bariatric surgery). If, on the other hand, we know that the increased appetite following weight loss does subside following a period of weight maintenance at the lower weight, or if we could identify those individuals for whom this is possible, then this information could be used to promote compliance with weight maintenance strategies after weight loss, with patient messages such as; "Keeping weight off will be difficult in the beginning, but don't give up because it will likely become easier with time".

A substantial body of research shows that a diet that is higher in protein (Westerterp-Plantenga et al 2004) and lower in glycaemic index (Brand-Miller et al 2002) than the diet that is conventionally recommended for health may help people to maintain a lower body weight post weight loss, and that this benefit may occur by reducing the drive to eat. These findings raise the possibility that a higher protein and lower glycaemic index weight maintenance diet could be used to reduce the intensity of the famine reaction in overweight or obese people after completion of weight loss diets, thereby improving their likelihood of keeping the weight off. While there is ample research showing the benefits of a higher protein diet for reducing appetite, enhancing weight loss and preserving lean body mass loss when applied during energy restricted weight loss programs, as recently reviewed (Soenen et al 2013), there is relatively little work to date about the emerging benefits of a higher protein diet applied during a weight maintenance program after weight loss. The importance of this Sub-Study (Part 1) is that it not only seeks to confirm a controversial new finding that has potentially enormous implications for the clinical management of overweight and obesity (Sumithran et al 2011), it also investigates the potential benefit of a higher protein and lower GI diet - applied during the weight maintenance phase after a standardized weight loss program - to prevent the apparently permanent increase in appetite that overweight and obese people have been reported in one study (Sumithran et al 2011) to experience in response to weight loss.

Part 1 Hypothesis:

• Both overweight and obese individuals will demonstrate increases in appetite and corresponding changes in appetite-regulating hormones in response to a standardized low calorie diet weight loss program, and these effects will be normalized within 4-10 months on a weight maintenance program in overweight but not in obese individuals.
• In both overweight and obese individuals, a higher protein and lower glycaemic index weight maintenance diet after a weight-reducing diet will improve normalisation of appetite and appetite-regulating hormones compared to a moderate protein and moderate glycaemic index weight maintenance diet.

Part 1 Aim:

• To determine fasting appetite and fasting circulating concentrations of appetite-regulating hormones (ghrelin and peptide YY) after a 2-month standardized low calorie diet in overweight and obese pre-diabetic adults, and to determine whether any effects are attenuated by 4 and 10 months on one of two different weight maintenance programs differing in protein content and glycaemic index.

PART 2. Effect of weight loss on bone homeostasis and muscle strength in younger and older overweight or obese pre-diabetic adults: impact of diet during weight maintenance on long-term durability of effects.

While it is generally recognised that losing excess weight helps to prevent disease development in younger adults, there is some controversy as to whether weight loss programs are indicated for the management of overweight or obesity in older adults, and if so, at what body mass index such programs should be implemented (Chapman 2008). It is noteworthy that weight loss via voluntary or involuntary means in older adults is linked to reduced function, reduced quality of life and increased mortality (Chapman 2008). The reason for this relationship is not clear, but one possibility is that weight loss programs can lead to significant loss of bone and lean tissues under certain circumstances (e.g. inadequate dietary protein, inadequate exercise), and it is unknown if these losses are recuperated. Given that reductions in bone density and lean body mass are risk factors for fractures and falls in older adults, changes in body composition in response to weight loss efforts in older adults could inadvertently produce negative effects. In light of the established role of dietary protein in the maintenance of lean tissues such as bone and muscle in older people or in response to weight loss (Westerterp-Plantenga et al 2012), the PREVIEW trial offers an invaluable opportunity to investigate the effects of a standardised weight loss program on bone homeostasis and muscle function (strength) in younger and older adults, as well as the impact of differences in diet during weight maintenance in attenuating any such effects.

Part 2 Hypothesis:

• That a 2-month standardized low calorie diet weight loss program leads to reductions in bone mass and muscle strength in both younger and older adults, particularly in older adults, and that these parameters return towards baseline values within 3 years in younger but not in older adults.
• That a higher protein weight-maintenance diet improves the restoration of bone mass and muscle strength after weight loss in both younger and older adults.

Part 2 Aim:

• To measure bone mass, bone turnover and muscle strength in younger (25-45 year old) and older (55-70 year old) adults before and after a standardized 2-month weight loss program, as well as at 6, 12, 24 and 36 months after commencement of the weight loss program.
• To compare the effects of two different weight maintenance programs differing in protein content on the restoration of bone homeostasis and muscle strength after a standardized 2-month weight loss diet. The weight maintenance programs are of 10 months' duration and are administered immediately after the 2-month weight loss program.

Inclusion Criteria:

• Age 25 - 45 years and 55 - 70 years
• Overweight or obesity status BMI>25 kg/m2
• Pre-diabetes. The criteria from WHO/IDF (International Diabetes Foundation) for assessing pre-diabetes will be used as the formal inclusion criteria, i.e. having: Impaired Fasting Glucose (IFG): Fasting venous plasma glucose concentration 5.6 - 6.9 mmol/l or Impaired Glucose Tolerance (IGT): Venous Plasma glucose concentration of 7.8 - 11.0 mmol/l at 2 h after oral administration of 75 g glucose (oral glucose tolerance test, OGTT), with fasting plasma glucose less than 7.0 mmol/l. Due to potential between-lab variation (local assessments), HbA1c is not used as an inclusion criteria in the screening.
• Informed consent required
• Ethnic group - No restrictions
• Smoking - Smoking is allowed, provided subjects have not recently (within 1 month) changed habits. However, smoking status is monitored throughout the study and used as a confounding variable.
• Motivation - Motivation and willingness to be randomized to any of the groups and to do his/hers best to follow the given protocol
• Other - Able to participate at CID's during normal working hours.

Exclusion Criteria:

Based on interview and/or questionnaire, individuals with the following problems will be excluded:

• Medical conditions as known by the subjects: Diabetes mellitus (other than gestational diabetes mellitus); Significant cardiovascular disease including current angina; myocardial infarction or stroke within the past 6 months; heart failure; symptomatic peripheral vascular disease; Systolic blood pressure above 160 mmHg and/or diastolic blood pressure above 100 mmHg whether on or off treatment for hypertension. If being treated, no change in drug treatment within last 3 months; Advanced chronic renal impairment; Significant liver disease e.g. cirrhosis (fatty liver disease allowed); Malignancy which is currently active or in remission for less than five years after last treatment (local basal and squamous cell skin cancer allowed); Active inflammatory bowel disease, celiac disease, chronic pancreatitis or other disorder potentially causing malabsorption; Previous bariatric surgery; Chronic respiratory, neurological, musculoskeletal or other disorders where, in the judgement of the investigator, participants would have unacceptable risk or difficulty in complying with the protocol (e.g. physical activity program); A recent surgical procedure until after full convalescence (investigators judgement); Transmissible blood-borne diseases e.g. hepatitis B, HIV; Psychiatric illness (e.g. major depression, bipolar disorder).
• Medication: Use currently or within the previous 3 months of prescription medication that has the potential of affecting body weight or glucose metabolism such as glucocorticoids (but excluding inhaled and topical steroids; bronchodilators are allowed), psychoactive medication, epileptic medication, or weight loss medications (either prescription, over the counter or herbal). Low dose antidepressants are allowed if they, in the judgement of the investigator, do not affect weight or participation to the study protocol. Levothyroxine for treatment of hypothyroidism is allowed if the participant has been on a stable dose for at least 3 months.
• Personal/Other: Engagement in competitive sports; Self-reported weight change of >5 % (increase or decrease) within 2 months prior to screening; Special diets (e.g. vegan, Atkins) within 2 months prior to study start. A lacto-vegetarian diet is allowed; Severe food intolerance expected to interfere with the study; Regularly drinking > 21 alcoholic units/week (men), or > 14 alcoholic units/week (women); Use of drugs of abuse within the previous 12 months; Blood donation or transfusion within the past 1 month before baseline or CID's; Self-reported eating disorders; Pregnancy or lactation, including plans to become pregnant within the next 36 months; No access to either phone or Internet (this is necessary when being contacted by the instructor's during the maintenance phase); Adequate understanding of national language; Psychological or behavioral problems which, in the judgement of the investigator, would lead to difficulty in complying with the protocol.
• Laboratory screening: If all of the above criteria are satisfied, the participant is eligible for a glucose tolerance test (blood at 0 and 120 mins), and blood glucose concentrations are analyzed immediately (Haemocue). In addition full blood count, urea, and electrolytes may be analyzed as a further safety evaluation.
• ONLY IF the glucose tolerance test meets the entry criteria for the study, the remaining samples are sent to the local laboratory for a safety check, with the following exclusion criteria: Hemoglobin concentration below local laboratory reference values (i.e. anemia); Creatinine >1.5 times Upper Limit of Normal (local laboratory reference values); Alanine Transaminase (ALT) and/or Aspartate Transaminase (AST) >3 times the Upper Limit of Normal (local laboratory reference values); Or any other significant abnormality on these tests which in the investigators opinion may be clinically significant and require further assessment.
• Electrocardiography (ECG). Any abnormality which in the opinion of the investigator might indicate undiagnosed cardiac disease requiring further assessment (e.g. significant conduction disorder, arrhythmia, pathological Q waves). This is done in adults 55-70 years of age.
• After LCD phase (in adults): Failure to reach at least 8% weight reduction during the LCD phase. This leads to exclusion from the intervention.