We previously reported the effects of transitioning from 4 weeks of a 15% protein, 50% carbohydrate, and 35% fat baseline diet (BD) followed immediately by 4 weeks of an isocaloric 15% protein, 5% carbohydrate, and 80% fat KD on energy expenditure in 17 men with overweight or class I obesity ( 7 ). We also explored the changes in glucose and lipid homeostasis and in inflammatory markers in this population to test the hypothesis that these comorbidity risk factors were significantly affected by dietary macronutrient content.

Very low‐carbohydrate, high‐fat, ketogenic diets (KDs) have become increasingly popular for the treatment of obesity and type 2 diabetes ( 1 ). However, little is known about the effects of transitioning people from a high‐carbohydrate diet to an isocaloric KD on glucose and lipid homeostasis or inflammation. Such very low‐carbohydrate diets have been reported in some studies to result in decreased energy intake (EI) ( 2 ), improved glucose and lipid homeostasis, and decreased inflammatory biomarkers ( 3 ). However, most of these studies have been in outpatients in whom it is difficult to disassociate physiological dietary effects from those related to dietary adherence ( 4 , 5 ), even in controlled feeding studies in which all food is provided ( 6 ). Free‐living diet studies do not investigate the effects of actually consuming diets but instead investigate the effects of the instructions to change diet in the prescribed way. To understand the metabolic effects of an isocaloric KD, an inpatient controlled feeding study is required.

Within‐subjects comparisons were made by ANOVA with repeated measures in which either diets (BD or KD) or MMTs (CM or KM) were used as covariates. To increase the likelihood that all biochemical values were “stable” within study periods, regression equations were calculated comparing values for fasting blood concentrations during the last 2 weeks of each diet. To determine whether measures obtained during the BD were predictive of those observed during the KD, regression equations were calculated comparing the mean values of fasting concentrations over the last 2 weeks of the BD with those during the last 2 weeks ingesting the KD. Analyses of the relationship of background diet and MMT type on the relationship of fasting measures of insulin sensitivity to measures derived from the MMT were made by multiple linear regression analysis in which the dynamic (MMT‐derived) measure was the dependent variable and the fasting measures, diet type, and meal type were each treated as dichotomous variables.

Adipose tissue sensitivity to insulin‐mediated changes in circulating FFAs (integrating simultaneous effects on lipolysis and esterification) was calculated from the Belfiore Index (Belfiore MMTFFA ) ( 18 ) and compared with results from the ADIPO‐IR, which is based on fasting data. Equations and additional calculations are presented in Table 2 .

There are few studies of the effects of isocaloric KDs on β‐cell function and insulin sensitivity or fasting versus postprandial assessments of these variables. To address these issues, fasting measurements were used to calculate the homeostatic model assessment (HOMA) of insulin resistance (IR) (primarily hepatic insulin sensitivity), HOMA‐β (insulin release), and adipocyte IR (ADIPO‐IR) ( 17 ), along with the fasting Belfiore Index ( 18 ). Glucose and insulin responses to MMTs were determined using postprandial relative AUCs. Absolute AUC glucose and AUC insulin were used in the calculation of the MMT Belfiore Index (Belfiore MMTglucose ) ( 18 ) normalized using the mean absolute values of AUC glucose and AUC insulin during the CM on the BD. The Matusuda Index—previously validated against clamp studies and in both oral glucose tolerance tests and MMTs ( 19 )—was also calculated as a postprandial index of insulin sensitivity. The Insulinogenic Index ( 20 ) and the Insulin Secretion‐Sensitivity Secretion Index 2 were chosen to assesses, respectively, β‐cell function using isolated time points (0 and 30 minutes) and the absolute AUC data, as well as uncorrected and corrected data for variations in insulin sensitivity ( 20 ) from MMT results.

In order to justify the examination of multiple variables relevant to glucose homeostasis without necessitating post hoc adjustments for multiple comparisons ( 9 ), assessments of areas under the curve (AUCs) relative to their fasting premeal concentrations for insulin (relative AUC insulin ) and glucose (relative AUC glucose ) during MMTs were assessed prior to analysis of other variables relevant to glucose homeostasis. Because there were significant dietary macronutrient effects on insulin and glucose during MMTs (see Results ), additional, more detailed analyses of glucose homeostasis were performed. Molecules subsequently studied were those that might mechanistically affect or reflect gluconeogenesis (e.g., glucagon) ( 10 ), insulin sensitivity (cortisol, FFA ( 11 , 12 ), and fibroblast growth factor 21 [FGF21] ( 13 )), insulinogenesis (gastric inhibitory peptide [GIP] ( 14 ), glucagon‐like peptide 1 [GLP‐1] ( 15 ), and peptide YY [PYY] ( 16 )), and inflammation (C‐reactive protein [CRP] and interleukin‐6 [IL‐6]) as well as relative AUCs for triglycerides (relative AUC triglyceride ), FFAs (relative AUC FFA ), and β‐hydroxybutyrate (relative AUC β‐hydroxybutyrate ). Similarly, analyses of cholesterol subfractions (high‐density lipoprotein [HDL] and low‐density lipoprotein [LDL]) and triglycerides were not made until a significant diet effect on total cholesterol was noted.

Data are presented as mean (SD). The primary outcome variables of these biochemical studies were those relevant to glucose homeostasis and circulating lipids, in contrast to earlier studies that focused primarily on energy expenditure ( 7 ).

Subjects underwent weekly fasting blood draws for assessment of ketosis, lipids, glucose and fatty acid homeostasis, and inflammation (Table 1 ). During the second week of EI stability on the BD (defined as week 4), subjects underwent 2 isocaloric mixed‐meal tests (MMTs) at ~9 am, at least 12 hours after their last meal. The test meal consisted of 20% of total daily prescribed energy with macronutrient distribution identical to the BD (designated as a “control meal” [CM]); and at least 3 days later, subjects received a meal of equal calories providing the same macronutrient distribution as the KD (designated as a “ketogenic meal” [KM]). Plasma and serum samples for glucose, insulin, free fatty acids (FFAs), β‐hydroxybutyrate, and triglycerides were obtained at −10, −5, 0, 5, 10, 15, 30, 45, 60, 90, and 120 minutes relative to completion of meal consumption. Subjects were then switched to an isocaloric KD (15% protein, 5% carbohydrate, 80% fat) for 4 weeks. During week 4 on the KD, subjects underwent MMT. The order of MMTs on the KD was KM first, followed at least 3 days later by the CM. Assays are described in online Supporting Information.

Diets consisted of 7‐day rotating menus using NUTRITIONIST PRO software (version 1.3; First Databank Inc., The Hearst Corp., San Bruno, California). The energy and macronutrient composition for each day was verified by chemical analysis (Covance Laboratories, Madison, Wisconsin). Food was prepared in the Pennington Biomedical Research Center metabolic kitchen, frozen, and shipped to the study sites, where meals were prepared for consumption according to standardized procedures and addition of fresh produce. Both menus contained minimal quantities of processed food, and despite the large differences in macronutrient composition, the ratios of refined to unrefined sugars were similar between the diets. This control of macronutrient quality permitted examination of the effects of the relative macronutrient contents without confounding due to differences in the types of nutrients (refined vs. unrefined sugars, simple vs. complex carbohydrates, saturated vs. mono‐ or polyunsaturated fatty acids) used. Quality of protein was monitored by using similar protein sources on corresponding BD and KD menu days (e.g., white‐meat chicken on the BD and dark‐meat chicken on the KD). Sample menus have been reported previously ( 7 ).

The study protocol was approved by the institutional review boards of the National Institute of Diabetes and Digestive and Kidney Diseases (ClinicalTrials.gov identifier NCT01967563), the Pennington Biomedical Research Center (2013‐3‐PBRC), Columbia University Medical Center (IRB‐AAAL7113), and the Translational Research Institute for Metabolism and Diabetes (FH IRB‐493675) and was consistent with guiding principles for research involving humans ( 8 ). Written informed consent was obtained from all subjects.

As previously reported ( 7 ), 17 men without diabetes and with BMI between 25 and 35 kg/m 2 were inpatients at 4 study sites (Table 1 ), where they resided on metabolic wards without access to food other than that provided within this study. Subjects spent 23 h/d for 2 consecutive days each week in a respiratory chamber. An initial weight maintenance energy requirement estimate was made as 1.5 times resting energy expenditure obtained by indirect calorimetry during the screening process. Subjects initially received the BD (15% protein, 50% carbohydrate, 35% fat), and caloric intake was further adjusted to achieve energy balance to within 5% of the average daily energy expenditure measured by the respiratory chamber based on the average of the two 23‐hour chamber stays during that week of the BD (range to completion: 2‐3 weeks). Subjects underwent 2 consecutive 23‐hour chamber stays on the same days each week throughout the study. After 4 weeks of EI stability on the BD, subjects were switched to an isocaloric KD (15% protein, 5% carbohydrate, 80%) for 4 weeks. The last 2 weeks of each diet were designated as the “test period” to allow subjects time to demonstrate persistent agreement between dietary intake and chamber calorimetry during the BD and to allow accommodation for the switch to the KD.

Correlational analyses between fasting and MMT‐derived indices of fuel use were examined for diet and meal effects (see online Supporting Information). We found highly significant correlations of fasting and dynamic indices of insulin sensitivity and FFA flux, but not insulin secretion, suggesting that extrapolations of fasting to postprandial measures of insulin‐mediated glucose disposal are dependent on both current diet and acute meal type.

Lipids during the control meal (CM) and ketogenic meal (KM) mixed‐meal tolerance tests. Free fatty acids (FFAs) and glycerol were significantly greater at all time points on the ketogenic diet (KD). Data are mean (SD). ( A ) FFA excursion during morning meal testing with the CM. ( B ) FFA excursion during morning meal testing with the KM. ( C ) Triglyceride excursion during morning meal testing with the CM. ( D ) Triglyceride excursion during morning meal testing with the KM. ( E ) Glycerol excursion during morning meal testing with the CM. ( F ) Glycerol excursion during morning meal testing with the KM.

Fasting total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides were all stable and significantly correlated between weeks 3 and 4 and within each diet (Figure 2 , Table 7 ). Fasting total and HDL cholesterol were significantly higher, and triglycerides were significantly lower during the KD. The KM led to increased relative AUC triglyceride , regardless of the prevailing diet, and the relative AUC triglyceride during the KM was increased on the KD. Fasting concentrations of total, HDL, and LDL cholesterol (but not triglycerides) on the BD were highly correlated with values during the KD.

The inflammatory cytokines (CRP and IL‐6), adiponectin, PYY, the insulinogenic and lipoprotein lipase‐activating GIP, and the insulinogenic GLP‐1 and FGF21 were stable and significantly correlated between weeks 3 and 4 and within each diet (Table 5 ). Circulating concentrations of CRP, adiponectin, glucagon, and GIP were significantly higher, whereas FGF21 was significantly decreased during the KD. PYY was not significantly changed. In all cases except FGF21, values during the BD were highly correlated with those measured during the KD.

With the exception of relative AUC glucose during the MMT on the BD, measurements of glucose and insulin concentrations and other molecules relevant to glucose and FFA homeostasis were highly correlated between BD and KD phases of the study.

During the KM, the relative AUC for both glycerol and triglycerides was higher during the KD (Table 6 ). Insulin‐mediated antilipolysis was reduced during the KD, whether calculated from fasting data (ADIPO‐IR) or based on the MMT (Belfiore MMTFFA ).

Absolute (Figure 1 ) and relative (Table 6 ) AUC C‐peptide values were significantly higher during the CM on the KD (both P < 0.0001), suggesting that the insulin differences during the CM between KD and BD were the result of increased insulin secretion rather than reduced clearance. Table 6 indicates that the AUC glucose relative to premeal values was significantly higher during both MMTs on the KD, and the relative AUC insulin was higher after the CM on the KD. The Belfiore MMTglucose Index indicated that the KD resulted in impaired insulin sensitivity during the CM test, whereas the Matsuda Index reflected improved insulin sensitivity during the KM test. Fasting tests of insulin sensitivity were not significantly different between diets. There were no significant effects of diet on any indices of insulinogenesis.

Results of morning mixed‐meal tests. Glucose, insulin, and C‐peptide excursions during the control meal (CM) with composition matched to the baseline diet (BD) (solid lines, solid markers) were significantly greater during the ketogenic diet (KD) than during the BD. After an isocaloric ketogenic meal (KM) (dashed lines, open markers), only the glucose excursion was significantly increased during the KD, and C‐peptide levels were significantly lower on the KD at 15 minutes and 120 minutes. *Diet difference, P < 0.05. See Methods for compositions of BD and KD. Data are mean (SD). ( A ) Glucose excursion during morning meal testing with a CM meal. ( B ) Glucose excursion during morning meal testing with a KM meal. ( C ) Insulin excursion during morning meal testing with a CM meal. ( D ) Insulin excursion during morning meal testing with a KM meal. ( E ) C‐peptide excursion during morning meal testing with a CM meal. ( F ) C‐peptide excursion during morning meal testing with a KM meal.

Fasting insulin, glucose, and C‐peptide values were stable and significantly correlated between weeks 3 and 4 and within each diet (Tables 4-6 ). We reported previously ( 7 ) that absolute insulin secretion, as estimated by 24‐hour urinary C‐peptide excretion, was significantly decreased during the KD. Figure 1 and Table 6 illustrate that the absolute and relative AUC glucose values were significantly higher during the CM and KM on the KD ( P = 0.0001) and that the absolute and relative AUC insulin , but not the AUC glucose , values were significantly higher during the CM on the KD ( P = 0.0002), suggesting that insulin sensitivity was decreased during the CM (higher glucose and higher insulin) and probably decreased (higher glucose despite no significant decline in insulin) during the KM in subjects during the KD.

Acetoacetate, β‐hydroxybutyrate, and glycerol concentrations were stable and significantly correlated between weeks 3 and 4 within each diet, indicating stable rates of ketosis. FFA values were significantly higher on week 4 of the BD compared with week 3 without significant interweek correlation on either diet. Plasma ketones, FFAs, and glycerol were all significantly higher during the last 2 weeks of the KD compared with the BD. Fasting plasma ketones, FFAs, and glycerol measured during the BD were not significantly correlated with those measured during the KD (Table 3 ).

Subjects (Table 1 ) were in a state of negative energy balance due to inadvertent underfeeding throughout the study. Weight decreased by 0.8 (0.2) kg ( P = 0.002) during the last 15 days of the BD and 0.2 (0.1) kg (not significant) during the last 15 days of the KD ( 7 ). Energy expenditure by chamber calorimetry was the primary outcome variable of this study and was approximately 60 kcal/d higher on the KD, predominantly because of early changes in energy expenditure ( 7 ).

Discussion

We examined biochemical parameters relevant to insulin sensitivity, inflammation, and dyslipidemia during consumption of a BD, whose relative composition of fat, carbohydrates, and protein was similar to the average American diet, and following the transition to an isocaloric KD as part of a study of the effects of diet composition on energy homeostasis (7). We found that switching from the BD to the KD resulted in significantly increased circulating concentrations of fasting ketones, glycerol, FFAs, glucagon, adiponectin, GIP, total and LDL cholesterol, and CRP and decreased fasting insulin, C‐peptides, triglycerides, and FGF21. MMTs indicated that the AUC glucose was significantly higher during the KD, regardless of whether the test meal was CM or KM, and the AUC insulin and AUC C‐peptide during the KD were significantly higher during the CM. Measures of glucose homeostasis derived from the MMT suggested that the KD impaired insulin sensitivity in response to the CM and improved sensitivity in response to the KM. Insulin‐mediated antilipolysis during the KD was decreased following both MMTs.

Similar to the authors of the present study, Jebb et al. (21) found no effects of isocaloric low‐glycemic‐index diets versus high‐glycemic‐index diets on insulin sensitivity over a 4‐week period. Partsalaki et al. (22) examined the effects of an ad libitum KD versus a low‐calorie diet (30%‐35% fat, 50%‐55% carbohydrate; 500‐kcal/d deficit) in children with obesity and found that although there was greater weight loss on the KD, there were no significant differences in insulin sensitivity beyond those attributable to weight loss. Some studies have reported significant adverse effects of overfeeding of high‐ versus low‐carbohydrate diets on insulin sensitivity during weight gain (23) and on hepatic insulin sensitivity during weight regain (24), suggesting that both weight change and diet composition significantly alter carbohydrate effects on glucose homeostasis. Other studies, some more recent (25) and others dating back more than 75 years (26), have reported that low‐carbohydrate diets, but not necessarily KDs, may result in impaired glucose tolerance in some individuals. The overall lack of a clear, generalizable health benefit of one diet over the other is similar to that recently reported by Gardner et al. in subjects studied over 12 months on low‐carbohydrate versus low‐fat weight loss diets (27).

The physiological significance of the apparent increased insulin sensitivity in response to a KD MMT versus the decreased insulin sensitivity in response to a BD MMT on the KD (both relative to the BD) cannot be determined from these data. The health consequences of improved insulin sensitivity as long as one remains on a KD may be small because of the consistently low circulating insulin concentrations on said diet. Similarly, the duration of the implied decreased insulin sensitivity in someone transitioning from a low‐carbohydrate diet or KD to a higher‐carbohydrate diet and the long‐term health effects cannot be assessed at present.

Blunting of insulin‐mediated antilipolysis on the KD should favor loss of body fat. However, the physiological significance of this finding is unclear because it seems unlikely that the additional effects of the KD to diminish insulin‐mediated antilipolysis would add significantly to the high levels of lipolysis already occurring as a result of low circulating insulin concentrations. In fact, loss of body fat was not increased during the KD period as compared with the BD period, as previously reported (7).

The increases in plasma adiponectin during the KD would presumably promote insulin sensitivity (28) but may reflect the state of negative energy balance and weight loss rather than an effect of reduced carbohydrate ingestion per se. Other studies of moderate carbohydrate restriction (< 30% total calories) without weight loss, with caloric restriction (−500 kcal/d for 4‐6 weeks, a greater negative energy balance and weight loss than in the present study) have not found a significant carbohydrate effect on circulating adiponectin (29). There are, to our knowledge, no studies of the effects of a weight maintenance KD on adiponectin in humans other than a study of 10 children with refractory epilepsy due to glucose transporter 1 deficiency, which found no significant changes in circulating adiponectin after 3 months of a KD (30).

Low‐protein diets in humans and mice have been shown to result in increased FGF21 (31), whereas calorie‐restricted KDs have been reported to decrease FGF21 (32). In this isocaloric study, as the protein content of the BD and KD remained constant, the observed decrease in FGF21 on the KD most likely reflected the KD (33). Reduced FGF21 could be associated with decreased glucose uptake by adipocytes during carbohydrate restriction, as has been reported in mice (13), and may be ameliorated by ketosis (34). Circulating concentrations of FGF21 on either diet were not significantly correlated with any fasting or MMT‐derived indices of insulin secretion or sensitivity.

A recent meta‐analysis (35) suggested that a KD during weight loss results in a small but significant decrease in appetite. Studies during weight maintenance using a low‐carbohydrate diet have yielded varied results (36, 37). The lack of differences between diets in in circulating concentrations of PYY and GLP‐1 suggests that any effects of a KD on appetite are not mediated by these molecules.

Circulating concentrations of CRP, but not the inflammatory cytokine IL‐6, were significantly higher on the KD. Ketosis in mice has been reported to increase tissue‐specific (liver and white adipose tissue) expression of inflammatory cytokines (e.g., tumor necrosis factor alpha, IL‐6, and macrophage markers (38)) and to downregulate the FGF receptor (39). Outpatient studies of low‐carbohydrate diets in humans have found significant increases in plasma CRP but not IL‐6 (5, 40). In subjects with type 2 diabetes, advice to follow a lower‐carbohydrate diet decreased IL‐6 but not CRP concentrations (5, 41).

During the KD, our subjects had higher fasting levels of plasma total and LDL cholesterol but had lower triglycerides. Interestingly, the AUC triglyceride values were higher during the KD regardless of the composition of the MMT, but the KM led to significantly greater postprandial triglyceride excursion (peak − baseline) on both diets. Furthermore, the AUC triglyceride after the KM was further increased during the KD period; this result differs from those of previous studies showing that adaptation to a high‐fat/low‐carbohydrate diet decreases postprandial triglycerides following a high‐fat meal (42). Perhaps the limited duration of the MMTs in the current study was not sufficient to reflect improvements in triglyceride clearance associated with adaptation to a high‐fat diet.

With the exception of triglycerides and FGF21, all BD measures of insulin sensitivity, molecules affecting glucose homeostasis, and plasma lipids were highly predictive of values obtained during the KD. These correlations suggest that although the rank order of propensity toward adiposity‐related comorbidities within a given population on a similar diet remains stable, the absolute risk of adiposity‐related comorbidities can be modified, but not eliminated, by diet modification.

A strength of this study is that it was conducted in an inpatient environment with assured dietary and behavioral compliance, as evident in biochemical/endocrine test stability. This investigation is limited by its examination of an extremely low‐carbohydrate (ketogenic) diet that differed only in relative macronutrient proportions, not macronutrient quality. The results cannot be extrapolated to low‐carbohydrate, nonketogenic diets; to diets with specific restrictions on types of sugars or fats; or to the effects of dietary macronutrient content on comorbidities over longer periods of time. The lack of a randomized crossover design fails to control for possible significant effects of testing order (5). The unintentional weight loss may have biased the results toward improved insulin sensitivity, lipids, and inflammatory markers in subjects on the KD (5).

In summary, this study shows that switching from a BD to an isocaloric KD is associated with the following: increased plasma LDL cholesterol but decreased triglycerides, increased plasma CRP but decreased FGF21, reduced glycemic insulin sensitivity to a meal with normal carbohydrate content, improved insulin sensitivity to a KM, and impaired antilipolysis insulin sensitivity regardless of meal type. Importantly, even drastic changes in diet macronutrient composition did not affect the rank order of an individual’s risk factors for metabolic disease. Those subjects with the highest and lowest risk reflected by glucose homeostasis, inflammation, and circulating lipid concentrations maintained the same relative rank order regardless of diet.