High-protein diets have been recently demonstrated to effectively reduce insulin resistance, derangements of the lipid profile and liver fat content in subjects with moderately and severely impaired glucose metabolism and non-alcoholic fatty liver disease (LeguAN, LEMBAS, DiNA-P, DiNA-D). The effects can be attributed to prolonged insulin secretion and improved second meal effect, higher energy expenditure by urea synthesis, suppression of glucagon or other mechanisms. Up to now, it is unclear, if proteins with slower or faster digestibility lead to differential results in these study designs. The proposed study will elucidate this question. The Investigators hypothesize, that slowly-digestible proteins induce a prolonged insulin plateau supporting the second-meal effect. The investigators also assume, that these dietary proteins lead to a markedly stronger short-term secretion of glucagon followed by desensitisation of this hormone release. Fast-digestible proteins, on the other hand, will presumably induce a smaller second-meal effect and do not inhibit a second rise of glucagon in a consecutive meal.

The investigators intend to study the effects of a 3-weeks high-protein diet in 80 subjects with NAFLD and T2DM on liver fat content (MR spectroscopy) and glucose metabolism. The investigators expect different results for slow protein (casein) and fast protein (whey), thus comparing both protein species. The two major clinical visits before and after the intervention period will include MRI spectroscopy, fasting blood sampling for later analysis, full anthropometric assessment, a mixed meal tolerance test and a set of behavioral tests, investigating decision making processes. In order to characterize the postprandial profiles (e.g. insulin, glucagon, amino acids) of the varying protein sources, preliminary meal tests are performed in overweight subjects with and without T2DM.

High-protein diets have been recently demonstrated to effectively reduce insulin resistance, derangements of the lipid profile and liver fat content in subjects with moderately and severely impaired glucose metabolism and non-alcoholic fatty liver disease (LeguAN, LEMBAS, DiNA-P, DiNA-D). The effects can be attributed to prolonged insulin secretion and improved second meal effect, higher energy expenditure by urea synthesis, suppression of glucagon or other mechanisms. Up to now, it is unclear, if proteins with slower or faster digestibility lead to differential results in these study designs. The proposed study will elucidate this question. The investigators hypothesize, that slowly-digestible proteins induce a prolonged insulin plateau supporting the second-meal effect. They also assume, that these dietary proteins lead to a markedly stronger short-term secretion of glucagon followed by desensitisation of this hormone release. Fast-digestible proteins, on the other hand, will presumably induce a smaller second-meal effect and do not inhibit a second rise of glucagon in a consecutive meal.

The investigators intend to study the effects of a 3-weeks high-protein diet in 80 subjects with NAFLD and T2DM on liver fat content (MR spectroscopy) and glucose metabolism. The investigators expect different results for slow protein (casein) and fast protein (whey), thus comparing both protein species. The two major clinical visits before and after the intervention period will include MRI spectroscopy, fasting blood sampling for later analysis, full anthropometric assessment, a mixed meal tolerance test and a set of behavioral tests to investigate decision making processes.

In order to characterize the postprandial hormonal and amino acid profiles (e.g. insulin, glucagon, amino acids) of the varying protein sources, preliminary meal tests are performed. The first tests assess the protein dose-finding in 20 participants, 10 with T2DM and 10 without. On each day of the dose-finding assessment pre-trial one of the following dosages is used in a single oral protein tolerance test (5 g, 10 g and 30 g of whey or casein each).The second tests assess whether 30 g mixes of whey and casein in variable proportions induce different hormonal profiles of glucagon and insulin in comparison with 30 g pea protein, served as drinks together with a standardized breakfast. Therefore, 20 subjects, 10 with Metabolic Syndrome and T2DM and 10 with Metabolic Syndrome without T2DM undergo seven separate investigation days. The third preliminary tests assess the role of the product matrix/consistency in 6 participants with overweight/obesity. Participants consume commercially available milk products each 30 g protein content (approx. 80% Casein) but with different product consistency on three separate investigation days. Subjects without prior diabetes diagnosis additionally undergo an initial oral glucose tolerance test (OGTT) to ensure healthy glucose levels.

All clinical assessments will be conducted in the Dept. Endocrinology, Diabetes and Nutrition, Charité, Campus Benjamin Franklin (Lead: Charité, A.F.H. Pfeiffer). Psychobehavioral tests (DIfE, Prof. Park), assessment of body fat distribution including liver fat (University Hospital Tuebingen, Dr. Machann) and measurements of amino acid levels throughout the meal tests (Technische Universität Berlin, Prof. Rohn) are secondary work packages.

Subcohort 1 (n=46):

Inclusion Criteria:

• healthy glucose levels or T2DM
• 40-79 years
• overweight/obesity

Main Exclusion Criteria:

• type 1 diabetes, prediabetes
• currently receiving treatment with insulin
• lactose intolerance, or food intolerance/allergy to any of the study products
• severe endocrine, gastrointestinal, metabolic, cardiovascular, pulmonary, inflammatory or psychiatric disorder
• active or recent relevant cancer
• intake of glucocorticoids or other medication that influences glucose metabolism
• pregnancy, breastfeeding

Subcohort 2 (n=80):

Inclusion Criteria:

• T2DM
• with NAFLD
• 18-79 years

Main Exclusion Criteria:

• type 1 diabetes, prediabetes
• currently receiving treatment with insulin
• lactose intolerance, or food intolerance/allergy to any of the study products
• severe endocrine, gastrointestinal, metabolic, cardiovascular, pulmonary, inflammatory or psychiatric disorder
• active or recent relevant cancer
• intake of glucocorticoids or other medication that influences glucose metabolism
• pregnancy, breastfeeding
• claustrophobia

For the preliminary meal tests all drinks and food supplements are provided in neutral bottles and cannot be identified by visual appearance, taste, texture or odour. Masking applied to participants, care providers, investigators and outcomes assessors.

For the interventional study provided drinks and food supplements were masked best possible for the participants.